General composition framework for ligand-controlled regulatory systems

ABSTRACT

The invention provides an improved design for the construction of extensible nucleic acid-based, ligand-controlled regulatory systems, and the nucleic acid regulatory systems resulting therefrom. The invention contemplates improving the design of the switches (ligand-controlled regulatory systems) through the design of an information transmission domain (ITD). The improved ITD eliminates free-floating ends of the switching and the competing strands, and localizes competitive hybridization events to a contiguous strand of competing and switching strands in a strand-displacement mechanism-based switch, thereby improving the kinetics of strand-displacement. The improved regulatory systems have many uses in various biological systems, including gene expression control or ligand-concentration sensing.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 12/228,665,filed on Aug. 14, 2008, which claims the benefit of the filing dateunder 35 U.S.C. § 119(e) to U.S. Provisional Application No. 60/966,398,filed on Aug. 28, 2007, the entire contents of the above-referencedapplications (including the specification and drawings) are incorporatedherein by reference.

STATEMENT REGARDING FEDERAL FUNDING

Work described herein was funded, in whole or in part, by Grant No.NIH-R21 GM074767-01A1 awarded by the National Institute of Health (NIH),and Grant No. DoD-W81XWH-06-1-0250 awarded by the United StatesDepartment of Defense (DoD). The United States Government has certainrights in this invention.

BACKGROUND OF THE INVENTION

Basic and applied biological research and biotechnology are limited bythe ability to get information into and from living systems and to acton information inside living systems. For example, there are only asmall number of inducible promoter systems available to provide controlover gene expression in response to exogenous molecules. Many of themolecular inputs to these systems are not ideal for broadimplementation, because they can be expensive and introduce undesiredpleiotropic effects. In addition, broadly applicable methods for gettinginformation from cells noninvasively have been limited to strategiesthat rely on protein and promoter fusions to fluorescent proteins, whichenable researchers to monitor protein levels and localization andtranscriptional outputs of networks, leaving a significant amount of thecellular information content currently inaccessible.

A striking example of a biological communication and control system isthe class of RNA-regulatory elements called riboswitches, comprisingdistinct sensor and actuation (gene-regulatory) functions, that controlgene expression in response to specific ligand concentrations. Buildingon these natural examples, engineered riboswitch elements have beendeveloped for use as synthetic ligand-controlled gene-regulatorysystems. However, research to date has largely focused on the generationof specific instances of RNA devices, which do not necessarily guideresearchers in how to translate that particular instance into otherinstances useful for specific systems or applications. Often times, theactivity of these RNA devices is dependent on the particular system inwhich it was developed, including sequences immediately surrounding theregulator element, and modularity is not maintained.

As a result, these early examples of switch engineering do not addressthe challenges posed above, because they lack portability acrossorganisms and systems, and their designs and construction do not supportmodularity and component reuse.

SUMMARY OF THE INVENTION

The invention described herein provides general design principles andmethods for implementing such principles, for constructingligand-regulated polynucleotide devices. These general design principlesestablish a framework to translate RNA engineering into a broadlyapplicable field, in that modular molecular platforms are employed toallow rapid, yet reliable construction of distinct ligand-regulatedpolynucleotide devices using a variety of RNA regulators which arefunctionally independent of the particular system in which they origin.The resulting ligand-regulated polynucleotide devices or gene-regulatorysystems can be used in a wide variety of biological systems to, forexample, control the expression of specific target genes in response tovarious effector molecules, or sense the presence/change ofconcentration of the effector molecules, and/or the change in status ofan environmental condition (pH, temperature, ion concentration, etc.).

Specifically, the invention provides five engineering design principles(DPs) in achieving this goal: DP1, scalability (a sensing platformenabling de novo generation of ligand-binding elements forimplementation within the sensor domain); DP2, portability (a regulatoryelement (such as ribozyme) that may be independent of cell-specificmachinery or regulatory mechanisms for implementation within theactuator domain, or a regulatory element that may depend on thecell-specific machinery but is largely independent of cell type); DP3,utility (a mechanism through which to modularly couple the controlsystem to functional level components); DP4, composability (a mechanismby which to modularly couple the actuator and sensor domains withoutdisrupting the activities of these individual elements); and DP5,reliability (a mechanism through which to standardize the transmissionof information from the sensor domain to the actuator domain).

The invention also provides a distinct or improved design for a stranddisplacement mechanism-based switch (ligand-controlled regulatorysystems), by utilizing an improved information transmission domain(ITD). The improved ITD eliminates free-floating ends of the switchingand the competing strands, and localizes competitive hybridizationevents to a contiguous strand of competing strand and switching strandin the switch, thereby improving the kinetics of strand-displacement.This design results in a distinct, and preferably improved stranddisplacement kinetics than other designs, such as certainstrand-slippage mechanism (or helix slipping internal mechanism) baseddesign used in the prior art.

Thus one aspect of the invention provides a polynucleotide comprising:(1) a modular actuator domain having one or more functional activities,wherein the modular actuator domain is not a ribozyme, (2) a modularsensor domain that detects concentration change of a molecule, or statuschange of an environmental condition (pH, ion concentration,temperature), and, (3) an information transmission domain between themodular actuator domain and the modular sensor domain, the informationtransmission domain comprising: (a) a general transmission region, (b) aswitching strand, (c) a competing strand, wherein the switching strandand the competing strand are in a continuous sequence and compete tobind to the general transmission region through hybridizationinteractions; wherein detection of the concentration or status change bythe modular sensor domain favors a conformation change in the modularactuator domain; wherein the conformation change is mediated by astrand-displacement mechanism in the information transmission domain tofavor the binding of the general transmission region to one of theswitching strand and the competing strand; and, wherein the conformationchange modulates the functional activity of the actuator domain.

Depending on the specific identity of the actuator domain, itsfunctional activity may include: the ability to serve as a substrate foran RNAi (RNA interference) pathway enzyme, such as Dicer or other RNaseIII enzyme; the ability to hybridize with a target nucleic acid andinhibit the expression of a protein encoded by the target nucleic acid;the ability to hybridize with a target nucleic acid and serve astemplate for nucleic acid amplification; the ability to modulatesplicing; and/or the ability to become a target sequence for siRNA(small interfering RNA), miRNA (microRNA), or antisense sequence, etc.Other functional activities of the various actuator domains aredescribed below in conjunction with the description of the variousactuator domains.

In a related aspect, the invention provides a method for rational designof a modular polynucleotide, the method comprising: (1) providing amodular actuator domain having one or more functional activities,wherein the modular actuator domain is not a ribozyme, (2) providing amodular sensor domain that detects concentration change of a molecule,or status change of an environmental condition (pH, ion concentration,temperature), and, (3) providing an information transmission domainbetween the modular actuator domain and the modular sensor domain, theinformation transmission domain comprising: (a) a general transmissionregion, (b) a switching strand, (c) a competing strand, wherein theswitching strand and the competing strand are in a continuous sequenceand compete to bind to the general transmission region throughhybridization interactions; wherein detection of the concentration orstatus change by the modular sensor domain favors a conformation changein the modular actuator domain; wherein the conformation change ismediated by a strand-displacement mechanism in the informationtransmission domain to favor the binding of the general transmissionregion to one of the switching strand and the competing strand; and,wherein the conformation change modulates the functional activity of theactuator domain.

In another related aspect, the invention provides a method for improvingthe design of a sensor-regulated polynucleotide, the polynucleotidecomprising: (1) an actuator domain having one or more functionalactivities, wherein the actuator domain is not a ribozyme, and, (2) asensor domain that detects concentration change of a molecule, or statuschange of an environmental condition (pH, ion concentration,temperature), the method comprising: providing an informationtransmission domain between the actuator domain and the sensor domain,the information transmission domain comprising: (a) a generaltransmission region, (b) a switching strand, (c) a competing strand,wherein the switching strand and the competing strand are in acontinuous sequence and compete to bind to the general transmissionregion through hybridization interactions; wherein detection of theconcentration or status change by the sensor domain favors aconformation change in the actuator domain; wherein the conformationchange is mediated by a strand-displacement mechanism in the informationtransmission domain to favor the binding of the general transmissionregion to one of the switching strand and the competing strand; and,wherein the conformation change modulates the functional activity of theactuator domain.

In certain embodiments, the polynucleotide comprises RNA, DNA, or acombination thereof.

In certain embodiments, the polynucleotide comprises one or moremodified nucleotides or nucleotide analogs.

In certain embodiments, the polynucleotide is single-stranded.

In certain embodiments, the modular actuator domain comprises anantisense sequence, an siRNA or precursor thereof, an miRNA or precursorthereof, an shRNA (short hairpin RNA) or precursor thereof, an RNase IIIsubstrate, an alternative splicing element, or an RNAi targetingsequence. For example, the conformational change may produce or removean intramolecular double-stranded feature in the actuator domain (whichincludes the RNase III substrate sequence), where the double-strandedfeature is the substrate for the extrinsic enzymatic activity, e.g., anRNase III enzyme activity. In certain cases, the RNase III enzyme isDicer or Drosha. In those embodiments, the substrate sequence isselected to produce siRNA, miRNA or a precursor or metabolite thereof inan RNA interference pathway, as a product of reaction with the RNase IIIenzyme. In a related embodiment, the substrate sequence forms apri-miRNA or pre-miRNA substrate for Drosha (i.e., localized in thenucleus), and the product of Drosha-mediated cleavage is an miRNA thatcan translocate to the cytoplasm and be acted on by Dicer, i.e., inputsinto the RNA interference pathway.

In another illustration, the conformation change can be one that altersthe ability of the substrate sequence to form an intermoleculardouble-stranded feature with a second (discrete) nucleic acid species,such as a target gene, where the double-stranded feature is thesubstrate for the extrinsic enzymatic activity. For instance, the secondnucleic acid species can be an mRNA, and the extrinsic enzymaticactivity alters the mRNA in a manner dependent on the formation of thedouble-stranded feature with the switch actuator domain, such as byactivation of an RNase H enzyme and/or RNase P enzyme or the like.

In still other examples, ligand binding to the aptamer can induce orprevent the substrate sequence from forming a substrate for such otherextrinsic enzymatic activities as polymerases, recombinases, ligases,methylases, glycosylases, or nucleases.

Similarly, the antisense sequence can be selected to alter the levels ofexpression of different splice variants through hybridization totranscripts in a manner that effects intron splicing.

In still other embodiments, the targeting sequence inhibits expressionof the target gene through homologous recombination with the targetgene, and becomes a substrate for recombinase in a manner dependent uponthe ligand-induced conformational change.

In certain embodiments, the actuator domain can be selected to hybridizeto an RNA transcript of the target gene and thereby reduce the amount ofprotein translated from the RNA transcript, and/or alter splicing of theRNA transcript. Alternatively, the targeting sequence can be selected tohybridize to a genomic sequence of the target gene and reduces theamount of RNA transcribed from the genomic sequence.

In certain embodiments, the modular actuator domain comprises asubstrate for RNase III, wherein the substrate, when processed by RNaseIII, produces an siRNA or miRNA that targets a transcript of the targetgene.

In certain embodiments, the subject switch includes a poly-adenylatetail, or in the case of the expression constructs, a coding sequencethat when transcribed, produces a poly-A tail on the switch transcript.It will be appreciated by those skilled in the art that the subjectswitch constructs can be derived from various nucleotides and nucleotideanalogs, as well as utilizing various linkage chemistries, such as maybe adapted for use in the present invention from the art of antisenseand siRNA constructs. To further illustrate, the switch can include oneor more non-naturally occurring nucleoside analogs and/or one or morenon-naturally occurring backbone linkers between nucleoside residues.Such analogs and linkers can be used to alter the stability, nucleasesusceptibility (or resistance) and/or bioavailability (such as cellpermeability) relative to a corresponding nucleic acid of naturallyoccurring nucleosides and phosphate backbone linkers.

In certain embodiments, the polynucleotide is functional in vivo.

In certain embodiments, the modular actuator domain has the one or morefunctional activities in vivo. For example, the functional activitiesmay comprise an ability to hybridize with a target polynucleotide, anability to be incorporated into a RISC complex to serve as an siRNA ormiRNA guide sequence, or an ability to be an RNase III substrate.

In certain embodiments, the modular sensor domain is an aptamer.

In certain embodiments, the modular sensor domain binds the molecule,preferably specifically. In certain preferred embodiments, ligandbinding and the resulting macro level conformation change is dependenton the dose or concentration of the ligand, though the does-responsecurve is not necessarily linear.

In certain embodiments, the molecule has a molecular weight of no morethan about 2000 Da, 1000 Da, 500 Da, 300 Da, 200 Da, 100 Da, or 50 Da.

In certain embodiments, the molecule/ligand is a small peptide, anucleic acid, a carbohydrate, a fatty acid or lipid, a non-peptidehormone (such as steroid), an amino acid or precursor thereof, anucleotide or precursor thereof, a vitamin, a metal ion, a metabolite, apost-translationally modified protein, a signal transduction secondmessenger, an enzyme co-factor, an enzyme substrate, a product of anenzyme-mediated reaction, or a metabolic precursor or product thereof.

Particularly for embodiments where it is intended that the subjectswitch be regulated using an ectopically administered ligand, the ligandis preferably one that is cell permeable.

In certain embodiments, the ligand can be a molecule produced by thecell. In other embodiments, the ligand can be a cell permeable agentthat is contacted with the cell, e.g., either by ectopic addition or bydiffusion from a neighboring cell.

Certain embodiments provide tissue or cell type-specific modulation ofthe concentration and/or activity of a ligand or the expression of atarget gene. The tissue or cell type-specific modulation may be achievedby the tissue or cell type-specific presence of the ligand. For example,the aptamer domain of an aptamer-regulated nucleic acid may beresponsive to a tissue or cell type-specific ligand and the effectordomain targets a ligand to modulate the concentration and/or activity ofthe ligand. In another aspect, the aptamer domain of anaptamer-regulated nucleic acid is responsive to a tissue or celltype-specific ligand and the actuator domain targets a target gene tomodulate the expression of the target gene.

In certain embodiments, the switching strand and the competing strandsubstantially do not overlap.

In certain embodiments, the switching strand and the competing strandhave substantially the same sequence.

In certain embodiments, the switching strand and the competing strandare in tandem. For example, the switching strand and the competingstrand may be directly linked with each other in the polynucleotide,optionally with a few linker nucleotides. The switching strand and thecompeting strand may or may not be mutually exclusive, and they mayoverlap by a few nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9nucleotides, for example).

In certain embodiments, the switching strand and/or the competing strandare each at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, or more nucleotides in length.

In certain embodiments, the conformation change increases the functionalactivities. In other embodiments, the conformation change decreases thefunctional activities.

In certain embodiments, the extent of the conformation change inresponse to ligand/molecule concentration or status change is amenableto (rational) adjustment/tuning.

For example, the adjustment/tuning may be achieved through rationalsequence modification in the information transmission domain (to affectstructure stability and/or to vary the energy barrier between the twoadoptable conformations). Alternatively, the adjustment/tuning iseffectuated by modifying base-pairing interactions among the generaltransmission region, the switching strand, and/or the competing strand.Alternatively, the adjustment/tuning is effectuated by changing thelength of the paring base-pairs at one or both ends of the duplex formedbetween the general transmission region and the switching strand, and/orthe duplex formed between the general transmission region and thecompeting strand. Alternatively, the adjustment/tuning is effectuated bychanging base-pairing complementarity. Alternatively, adjustment/tuningis effectuated by changing the binding affinity between the modularsensor domain and the molecule without changing the size of the modularsensor domain. Alternatively, the adjustment/tuning is effectuated bychanging the size of the modular sensor domain. Depending on specificmechanisms of action of the actuator domains, this may alter theprocessing efficiency of the polynucleotide (e.g., when the actuatordomain is an RNase III substrate), the ability of an antisense actuatordomain to bind its target sequence, or the cleavage activity of aribozyme actuator domain, etc.

In certain embodiments, the extent of the conformation change inresponse to ligand/molecule concentration or status change is amenableto (rational) adjustment/tuning effectuated by any one or more of themechanisms described above.

Thus certain embodiments provide methods of designing, selecting, oroptimizing aptamer-regulated nucleic acids or aptamer domains that areresponsive to one or more pre-selected or pre-determined ligands. Suchswitches may also be “tuned” so that their switching behavior is more orless responsive to ligand binding. The switches may also be “tuned” sothat the binding affinity of the aptamer domain is more or lesssensitive to its ligand. For instance, the thermodynamic properties ofintramolecular duplex formation and other 2° and 3° structures in thesubject switch may be altered so that the aptamer domain is more or lessamenable to ligand binding, i.e., such as may be manifest in thedissociation constant (K_(D)) or other kinetic parameters (such asK_(on) and K_(off) rates). Alternatively, allosteric changes in theeffector domain may be more or less responsive to ligand binding uponalterations in hybridization and other intramolecular interactions thatmay effect 2° and 3° structures of the subject switch. Forwardengineering strategies for altering the thermodynamic properties ofnucleic acid structures are well known in the art. For instance,increased complementary nucleic acid pairing may increase the stabilityof an actuator or aptamer domain. It is anticipated that the absoluteand relative stabilities of the actuator domain and the aptamer domainwill be important design parameters in tuning the switch behavior of asubject switch.

In certain embodiments, the modular actuator domain can be(reliably/predictably) exchanged for a different modular actuator domainwithout substantially affecting the function of the modular sensordomain and the information transmission domain.

In certain embodiments, the modular sensor domain can be(reliably/predictably) exchanged for a different modular sensor domainwithout substantially affecting the function of the modular actuatordomain and the information transmission domain.

The invention also relates to a vector or expression construct encodingany of the subject polynucleotides.

In certain embodiments, the vector or expression construct may furthercomprise one or more transcriptional regulatory sequences that regulatetranscription from the vector or expression construct in a cellcontaining the vector or expression construct.

In certain embodiments, the cell is a mammalian cell, a human cell, arodent cell, a yeast cell, an insect cell, a worm cell, or a bacterium.

The invention also relates to a cell engineered to include any of thesubject polynucleotides, or any of the subject vector or expressionconstructs.

In other aspects, the invention provides a cell comprising: a metabolicpathway of one or more reactions that are regulated at least in part bya target gene; and one or more subject polynucleotides that act ascontrol elements on the metabolic pathway by regulating expression ofthe target gene through the modular actuator domain, wherein binding ofthe molecule to the modular sensor domain (e.g., aptamer) causes achange in the intramolecular interaction of information transmissiondomain, such that there is a change in the regulation of the target geneby the modular actuator domain, at a rate dependent upon the presence orabsence of the molecule.

Each of the subject polynucleotide can include (i) an aptamer sequencethat selectively binds to a ligand selected from an enzyme co-factor, areactant, a substrate or a product of a reaction in the metabolicpathway, (ii) an actuator domain, such as a gene silencing sequence forreducing expression of a target gene encoding a protein involved in themetabolic pathway. These may be proteins that act as enzymes in thepathway, or may be proteins that act as regulatory subunits or haveother effects on the pathway (such as transcription factors orrepressors that control expression of components of the metabolicpathway). In these embodiments, ligand binding to the aptamer causes achange in the regulated nucleic acid between two conformation states, inone of which the regulated nucleic acid inhibits expression of thetarget gene in a manner dependent on the gene silencing sequence, and inthe other of which the regulated nucleic acid does not inhibitexpression of the target gene. Thus, the metabolic pathway can beregulated at least in part by the regulated nucleic acid. In certainpreferred instances, the metabolic pathway includes at least onereaction mediated by an enzyme, and at least one of the regulatednucleic acid regulates expression of the enzyme.

Another aspect of the invention provides a library of aptamer-regulatednucleic acids, such as libraries having a variegated population ofnucleic acids having different aptamers and/or different actuatorregions (such as substrate sequences, antisense sequences or targetingsequences as described above). These libraries may have diversity amongthe aptamers with respect to the types of ligands that can be bound(specificity) and/or the variation in affinity for the same ligand.

In other aspects, the invention provides a method for regulatingexpression of a recombinant gene, comprising: (i) providing a cellengineered to include any of the subject polynucleotides, or any of thesubject vector or expression constructs, (ii) contacting the cell withthe molecule in an amount that alters the activity of the modularactuator domain. Preferably, the cell is contacted with themolecule/ligand in an amount that alters the activity of the modularactuator domain to a desired level. The relationship between theactivity of the actuator domain and the input molecule concentration maybe titrated in vitro or in vivo. The resulting titration curve may beused to provide precise regulation of actuator activity with specificligand concentration.

Another aspect of the invention provides a method for renderingexpression of a target gene in a cell dependent on the presence orabsence of a molecule, comprising introducing into the cell a subjectpolynucleotide comprising a modular actuator domain comprising asubstrate for RNase III, wherein the substrate, when processed by RNaseIII, produces an siRNA or miRNA that targets a transcript of the targetgene, wherein, binding of the molecule to the modular sensor domain(e.g., aptamer) causes a change in the intramolecular interaction of theinformation transmission domain, such that the substrate is processed byRNase III to produce the siRNA or miRNA to target the transcript, at arate dependent upon the presence or absence of the molecule.

In certain embodiments, the molecule is produced by the cell.

In certain embodiments, the molecule is a cell permeable agent that iscontacted with the cell.

Another aspect of the invention provides a method of determining theamount of an analyte in a cell which expresses a reporter gene,comprising: (1) introducing into the cell a subject polynucleotidecomprising a modular actuator domain comprising a substrate for RNaseIII, wherein the substrate, when processed by RNase III, produces ansiRNA or miRNA that targets a transcript of the reporter gene, whereinbinding of the analyte to the modular sensor domain (e.g., aptamer)causes a change in the intramolecular interaction of the informationtransmission domain, such that the substrate is processed by RNase IIIto produce the siRNA or miRNA to inhibit expression of the reportergene, at a rate dependent upon the presence or absence of the analyte;(2) measuring the amount of expression of the reporter gene; and (3)correlating the amount of expression of the reporter gene with theamount of analyte, thereby determining the amount of the analyte in thecell.

Another aspect of the invention provides a method for treating orpreventing infection by a pathogenic agent, comprising administering toa patient a sufficient amount of a subject polynucleotide, wherein themolecule is produced as a consequence of infection by the pathogenicagent, and wherein the modular actuator domain inhibits the function ofone or more genes essential for successful infection (e.g., maintenance,replication, or spread of pathogenic infection) by the pathogenic agent.Specifically, binding of the ligand to the aptamer domain favors aconformational change in the nucleic acid that alters the ability of theactuator domain to inhibit the expression of the target gene. Thenucleic acid is administered in a sufficient amount to inhibit theexpression of pathogen and/or host genes that are important for themaintenance, replication, or spread of pathogenic infection. Forexample, the aptamer domain of an aptamer-regulated nucleic acid canbind and respond to infection products (e.g., HIV gag, p24, p6, p7, p17,gp120, gp41, pol, env, tat, rev, nef, vif, vpr, vpu, and tev proteins)that are generated upon pathogen infection. Ligand binding to theaptamer domain favors a conformational change in the nucleic acid sothat the actuator domain is available to target pathogen and/or hostgenes that are important for the maintenance or spread of pathogenicinfection. Pathogens include, viral, eukaryotic and prokaryoticorganisms, including pathogenic viruses, bacteria, and fungi.

Still another aspect of the present invention provides a method ofconducting a pharmaceutical business comprising: (a) identifying asubject aptamer-regulated nucleic acid which, depending on specific use,when switched “on” (e.g., when the actuator domain becomes functional orhas an increased functional activity) or “off” (e.g., when the actuatordomain becomes non-functional or has a decreased functional activity)inhibits proliferation of target cells in vivo and reduces the effectsof a disorder involving unwanted proliferation of the target cells; (b)conducting therapeutic profiling of the aptamer-regulated nucleic acididentified in step (a) for efficacy and toxicity in animals; and (c)formulating a pharmaceutical preparation including one or more of theaptamer-regulated nucleic acids identified in step (b) as having anacceptable therapeutic profile.

The method of conducting a pharmaceutical business may further comprisean additional step of establishing a distribution system fordistributing the pharmaceutical preparation for sale, and optionally,establishing a sales group for marketing the pharmaceutical preparation.

Yet still another aspect of the present invention provides a method ofconducting a pharmaceutical business comprising: (a) identifying asubject aptamer-regulated nucleic acid which, when switched “on,”inhibits proliferation of target cells in vivo and reduces the effectsof a disorder involving unwanted proliferation of the target cells; (b)(optionally) conducting therapeutic profiling of an aptamer-regulatednucleic acid identified in step (a) for efficacy and toxicity inanimals; and (c) licensing, to a third party, the rights for furtherdevelopment of the aptamer-regulated nucleic acid.

The skilled artisan recognizes that an aptamer-regulated nucleic acidthat is useful for treating any disorder, including, but not limited toinhibiting pathogenic replication and/or infection, regulation of theimmune response, or modulation of the cellular state of a cell, may beused in the methods of conducting a pharmaceutical business.

Another aspect of the invention provides a method for causing phenotypicregulation of cell growth, differentiation or viability in cells of apatient, comprising introducing into cells in the patient a subjectpolynucleotide, where the modular sensor domain (e.g., aptamer) binds tothe molecule, the concentration of which is dependent on cellularphenotype, wherein binding of the molecule to the modular sensor domainfavors a conformational change that increases (or decreases) thefunctional activities of the modular actuator domain, and the increasedor decreased functional activities of the modular actuator domainmodulates expression of a target gene essential for altering theregulation of cell growth, differentiation or viability in the cells.

Merely for illustration, the method can be used to prevent the growth ofhyperplastic or tumor cells, or even the unwanted proliferation ofnormal cells. It can be used to induce the death of fat cells. It canalso be used to regulate growth and differentiation of stem cells, or toregulate activation of an immune response.

In certain embodiments, the method may be used to induce cell death in amanner dependent on the presence of the molecule.

In certain embodiments, the method is used to prevent cell death in amanner dependent on the presence of the molecule.

In certain embodiments, the method may be used to induce differentiationin a manner dependent on the presence of the molecule.

In certain embodiments, the method may be used to inhibitdifferentiation in a manner dependent on the presence of the molecule.

In certain embodiments, the method is used to prevent the growth ofhyperplastic or tumor cells.

In certain embodiments, the method is used to reduce fat cells in thepatient.

In certain embodiments, the method is used to regulate growth anddifferentiation of stem cells.

In certain embodiments, the method is used to regulate activation of animmune response.

In certain embodiments, the polynucleotide, or an expression constructfor transcribing the polynucleotide, are introduced ex vivo into cellswhich are transplanted into the patient.

In certain embodiments, the aptamer-regulated nucleic acid is introducedto cells, in vivo, by contacting the cells with an expression vectorhaving a nucleic acid coding sequence that is transcribed to produce oneor more products that produce the aptamer-regulated nucleic acid in thetreated cells. In other embodiments, the aptamer-regulated nucleic acidis introduced to cells ex vivo. For example, the aptamer-regulatednucleic acid may be introduced to cells outside of a subject bycontacting the cells with an expression vector having a nucleic acidcoding sequence that is transcribed to produce one or more products thatproduce the aptamer-regulated nucleic acid in the treated cells. Thecells that are transfected (e.g., stably transfected) with theaptamer-regulated nucleic acid can then be introduced into a subject fortreatment. The cells used in ex vivo treatment strategies may be derivedfrom the subject to be treated, from a donor, or from a previouslygenerated stock of maintained cells. Cells that may be used in ex vivotreatment strategies include, but are not limited to, stem cells,somatic cells, and immune cells (e.g., T cells). In a specificembodiment, the invention provides a method for modulating thedifferentiation of a stem cell, comprising transfecting a stem cell withan aptamer-regulated nucleic acid of the invention, wherein theaptamer-regulated nucleic acid comprises an aptamer domain and anactuator domain (e.g., an effector RNA domain). The aptamer domain isresponsive to the binding of a ligand and the effector domain istargeted to a molecule or gene that is sufficient to modulate thedifferentiation of a stem cell. Stem cells may be differentiated intoany cell type (e.g., a dermal cell, a hepatocyte, a retinal cell, etc.).

Another aspect of the invention provides a pharmaceutical preparationcomprising a subject polynucleotide, or an expression construct which,when transcribed, produces an RNA including the polynucleotide, and apharmaceutically acceptable carrier suitable for use administration to ahuman or non-human patient.

Another aspect of the invention provides a method of using the sequenceof an shRNA switch to facilitate the prediction of the relativeexpression level of a target gene (f_(model)) in vivo at the presence ofthe shRNA switch, the method comprising: (1) using an RNA secondarystructure prediction algorithm to provide a first active conformationand a second inactive conformation of the shRNA switch, based on thesequence of the shRNA switch; (2) using a stem method to calculate thefree energy difference (ΔG_(method)) between the inactive conformationand the active conformation, wherein the stem method only accounts forthe energetics of the major stem(s) in each conformation; (3)calculating the partitioning coefficient between the active and theinactive conformations (K_(Comp)) based on ΔG_(method) obtained in (2);(4) providing a predicted relationship between the relative expressionlevel of target gene and ligand concentration based on K_(Comp) obtainedin (3); and, (5) outputting the predicted relationship to a user on anoutput device, or verifying the predicted relationship with in vitroand/or in vivo experiments at one or more ligand concentration(s),wherein the shRNA switch comprises: (a) a duplex stem region that can beprocessed to siRNA or miRNA to antagonize the expression of the targetgene; (b) an aptamer that can bind a ligand over a range of ligandconcentrations, wherein the aptamer is within the loop region of theshRNA; (c) a switching strand within the loop region of the shRNA, andbetween the duplex stem region and the aptamer, wherein in the firstactive conformation, the aptamer does not bind the ligand, and theduplex stem region can be processed to the siRNA or miRNA; wherein inthe second inactive conformation, the aptamer binds the ligand andstabilizes the second inactive conformation, and the switching stranddisrupts the duplex stem region via a strand-displacement mechanism toabolish processing of the duplex stem region to the siRNA or miRNA.

In certain embodiments, suitable output device includes any suitabledisplay, such as computer monitor, any printed material, any data medium(CD, DVD, SD, memory card, hard drive, magnetic tape, etc).

In certain embodiments, in step (3):

$\begin{matrix}{K_{Comp} = {\sqrt[h]{{\frac{e \cdot f_{shRNA}}{C_{1}}\left\lbrack {C_{2} + {\exp \left( {- \frac{\Delta \; G_{method}}{k_{B}N_{A}T}} \right)}} \right\rbrack}^{C_{3}}} - 1.}} & (5)\end{matrix}$

wherein:

e is the processing efficiency of the duplex stem region to siRNA ormiRNA by in vivo RNAi pathway, and is calculated from the basalexpression levels from several related shRNA switches differing only inthe switching strand sequence and strongly preferring the first activeconformation;

f_(shRNA) is an experimentally obtained value of the relative knockdownof target gene expression by a corresponding shRNA having the sameduplex stem region but without the aptamer and the switching strand;

C₁-C₃ are fit constants obtained from fitting ΔG_(method) and the basalexpression levels for the several related shRNA switches using aleast-square analysis of

$\begin{matrix}{{f_{fit} = {1 - {C_{1}\left\lbrack {C_{2} + {\exp \left( {- \frac{\Delta \; G_{method}}{k_{B}N_{A}T}} \right)}} \right\rbrack}^{- C_{3}}}},} & {(2),}\end{matrix}$

wherein f_(fit) is the basal expression of the target gene for the fitcurve of Eq. (2);

k_(B) is the Boltzmann constant;

N_(A) is Avogadro's number;

T is temperature (in K); and,

h is the Hill coefficient that accounts for non-linearity between siRNAconcentration and the relative target gene expression level, and isobtained by generating a ligand response curve with one shRNA switchhaving negligible knockdown of the target gene expression at the highestligand concentration, and best fitting the curve with a least-squareanalysis.

In certain embodiments, the predicted relationship is represented by:

$\begin{matrix}{F_{model} = {1 - {e \cdot {\quad {{f_{shRNA}\begin{bmatrix}{1 + \left\lbrack {\sqrt[h]{{\frac{e \cdot f_{shRNA}}{C_{1}}\left\lbrack {C_{2} + {\exp \left( {- \frac{\Delta \; G_{method}}{k_{B}N_{A}T}} \right)}} \right\rbrack}^{C_{3}}} - 1} \right\rbrack} \\\left( {1 + {K_{Apt} \cdot L}} \right)\end{bmatrix}}^{- h},}}}}} & (6)\end{matrix}$

wherein:

K_(Apt) is the association constant between the ligand and the aptamer;

L is ligand concentration;

e is the processing efficiency of the duplex stem region to siRNA ormiRNA by in vivo RNAi pathway, and is calculated from the basalexpression levels from several related shRNA switches differing only inthe switching strand sequence and strongly preferring the first activeconformation;

f_(shRNA) is an experimentally obtained value of the relative knockdownof target gene expression by a corresponding shRNA having the sameduplex stem region but without the aptamer and the switching strand;

C₁-C₃ are fit constants obtained from fitting ΔG_(method) and the basalexpression levels for the several related shRNA switches using aleast-square analysis of

$\begin{matrix}{{f_{fit} = {1 - {C_{1}\left\lbrack {C_{2} + {\exp \left( {- \frac{\Delta \; G_{method}}{k_{B}N_{A}T}} \right)}} \right\rbrack}^{- C_{3}}}},} & {(2),}\end{matrix}$

wherein f_(fit) is the basal expression of the target gene for the fitcurve of Eq. (2);

k_(B) is the Boltzmann constant;

N_(A) is Avogadro's number;

T is temperature (in K); and,

h is the Hill coefficient that accounts for non-linearity between siRNAconcentration and the relative target gene expression level, and isobtained by generating a ligand response curve with one shRNA switchhaving negligible knockdown of the target gene expression at the highestligand concentration, and best fitting the curve with a least-squareanalysis.

In certain embodiments, in step (2), (i) the major stem for the firstactive conformation spans the entire duplex stem region, and, (ii) themajor stem for the second inactive conformation includes base-pairsformed between the switching strand and nucleotides in the duplex stemregion, excluding common regions in (i) and (ii).

In certain embodiments, the three-parameter curve fit of the ΔG_(method)values calculated with the stem method and the relative basal expressionlevels of the target gene has a coefficient of determination (R²) of atleast about 0.80 or 0.90.

The embodiments and practices of the present invention, otherembodiments, and their features and characteristics, will be apparentfrom the description, figures and claims that follow, with all of theclaims hereby being incorporated by this reference into this Summary.

It is contemplated that any embodiments described herein, includingthose only described under one of the many aspects of the invention, canbe combined with any other embodiments described under any aspects ofthe invention whenever appropriate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows design and characterization of an exemplary shRNA switchplatform. The shRNA stem (or actuator domain, shown as thedouble-stranded region with a 2-nt bulge in the middle of one strand),the aptamer domain (shown as the outlined stem-loop region in the toppart of the polynucleotide in FIG. 1A), and the switching strand (shownas the outlined sequence in the loop part of the polynucleotide in FIG.1A) are outlined. The competing strand (not outlined) overlaps with partof the actuator domain in the example shown in FIG. 1A. (A) Sequence andstructure of shRNA switch S1 and proposed mechanism for ligand controlof RNAi-mediated gene silencing. K_(Comp), K_(Apt), and e are parametersfrom the mathematical model; L denotes ligand. (B) In-line probing ofS4t under the following theophylline concentrations (μM): 0.001, 0.01,0.1, 1, 10, 100, 1000, and 8000. S4t was also resolved as unreacted(NR), partially digested with the G-specific RNase T1 (T1), and underbasic conditions (OH). The included secondary structure of S4t isrepresentative of the inactive conformation. Band quantification (right)is aligned with the resolved gel image. Nucleotides undergoing constant(medium gray shade), increased (light gray shade), or decreased (darkgray shade) cleavage in the presence of theophylline are shown. (C)Sequence and structure of shRNA switch S1 and associated controls. (D)Component transfer functions of S1 and switch controls. Dependence ofGFP levels on theophylline concentration for HEK293T tTA-d2EGFP cellstransfected with plasmids harboring the indicated constructs in thepresence of varying theophylline concentrations. Median fluorescencevalues from flow cytometry analysis were normalized to that ofuntransfected cells in the same well. Error bars represent one standarddeviation from duplicate transfected wells.

FIG. 2 is a model that predicts tuning of the shRNA switch transferfunction through variation of identified tuning parameters. Modelpredictions for the effect on the component transfer function of varyingK_(Comp) (A), K_(Apt) (B), or e (C). (D) Effect of e on the dependenceof basal expression levels on K_(Comp). Lines designate the minimalbasal expression set by f_(shRNA) (−) and the transfer function thatfits the S1 theophylline-response curve from FIG. 10 (−): K_(Comp)=0.17,K_(Apt)=0.016 μM⁻¹, e=0.85, f_(shRNA)=0.94, and h=1.33.

FIG. 3 shows experimental validation of switching strand tuningstrategies. (A) Designated strategies for physical modulation of thetuning parameters. Three strategies pertain to the switching strand(light gray) and reflect changes in K_(Comp), and two strategies pertainto the aptamer domain (dark gray) and reflect changes in K_(Apt) and e.(B-G) Tuned theophylline response curves as described in FIG. 1D andassociated RNA sequences. Each family of curves represents iterativenucleotide modifications under a single tuning strategy within thecompeting strand: 3′ end (B), 5′ end (D), and complementarity to theshRNA stem (F). Indicated sequence variants are swapped into theequivalent box in (A), which designates the applied tuning strategy foreach family of curves. Error bars represent one standard deviation fromduplicate transfected wells.

FIG. 4 shows experimental validation of aptamer tuning strategies. (A)Theophylline aptamer variants swapped into the equivalent box in FIG.3A. Dissociation constants (K_(D)) as previously reported (Zimmermann etal, 2000) are indicated for each aptamer. (B) Tuned theophyllineresponse curves as described in FIG. 1D for shRNA switches thatincorporate aptamers from (A). (C) Relationship between aptamer size andthe lower limit of basal expression levels estimated by shRNA switchesprimarily adopting the active conformation (see Materials and Methodsfor Examples 1-7). HEK293T tTA-d2EGFP cells were transfected withswitches containing the following aptamer: none (−), xanthine aptamer(xa), smaller theophylline aptamer (th_(S)), larger theophylline aptamer(th_(L)), or tetracycline aptamer (tc). The original shRNA targetingEGFP (sh) represents the lower theoretical limit in this cellularcontext. Values represent the average of at least one switch for eachaptamer. (D, E) Modular replacement of aptamer imparts new liganddependence while maintaining switch functionality. Hypoxanthine responsecurves were generated for shRNA switches incorporating the xanthineaptamer as described in FIG. 1D, except cells were grown in the presenceof varying concentrations of hypoxanthine. Indicated sequence variantsare swapped into the equivalent box in FIG. 3A. (F,G) Preservation ofswitching strand tuning strategies for shRNA switches containing thexanthine aptamer. Variation targeted the length of the 3′ end of theswitching strand. Error bars represent one standard deviation fromduplicate transfected wells.

FIG. 5 shows exemplary programming of transfer functions throughcombinatorial design strategies. (A) Combinatorial tuning strategiesenable fine tuning of the component transfer function. Step-wisenucleotide changes were made to S4, where each change fell under adifferent switching strand tuning strategy. (B) Tuned theophyllineresponse curves as described in FIG. 1D. Arrows depict the systematicmodifications designated in (A).

FIG. 6 is an extended model that enables sequence-to-transfer functionprediction and guides the forward design of optimized shRNA switches.(A) General process to convert shRNA switch sequence information into apredicted transfer function. RNA secondary structure algorithms and themethod displaying the highest correlation strength (stems method; andFIG. 11) were used to calculate the free energy difference betweenactive and inactive conformations (ΔG_(method)). This value issubsequently used to calculate K_(Comp), which is inserted into theextended model to yield the predicted relationship between ligandconcentration and target gene expression levels. (B) Predictedrelationship between basal expression levels and calculated free energydifference (ΔG_(model)) between active and inactive conformations. (C)Sequence-function relationship for shRNA switches under the stemsmethod. This method links sequence information to basal expressionlevels with the aid of RNA secondary structure prediction algorithms. ΔGwas calculated (ΔG_(method)) according to this method for shRNA switchsequences S1-10 and plotted with the associated measured basalexpression levels. The strength of the three-parameter curve fit wasevaluated based on the coefficient of determination (R²). Each datapoint represents one shRNA switch. (D) Extended model predictions forthe relationship between ΔG_(method) and dynamic range (η). η is definedas the ratio of GFP(%) at high (3 mM) and low (1 μM) theophyllineconcentrations. Curves represent shRNA switches containing the smallertheophylline aptamer (dark line; e=0.94, K_(Apt)=0.015 μM⁻¹) or thelarger theophylline aptamer (light line; e=0.85, K_(Apt)=0.016 μM⁻¹),respectively. (E) Values of η for shRNA switches containing the largertheophylline aptamer (S1-10; ∘) or the smaller theophylline aptamer(S11-25; ▪) as a function of ΔG_(method). Each data point represents oneshRNA switch. S13 (the optimized shRNA switch) and S1 (the originalshRNA switch) are marked. (F,G) Flow cytometry data for HEK293TtTA-d2EGFP cells transfected with S1 (F) or S13 (G) in the presence (,filled) or absence (∘) of 3 mM theophylline. Histograms fromuntransfected cells (□) or cells transfected with the original shRNAtargeting EGFP () are included.

FIG. 7 shows theophylline dependence of in-line cleavage at C56 () andC74 (∘) of S4t. Individual bands from the in-line gel (FIG. 1B) werequantified and normalized to an adjacent constant region to remove biasfrom inconsistent well loading. C56 bands were normalized to U47 bands,while C74 bands were normalized to U100-G102 bands. Curves were fitusing a standard Michaelis-Menten model, with determined K_(D) values of3.6 μM and 6.8 μM for C56 and C74, respectively.

FIG. 8 shows that antisense inhibition of guide strand activityrepresses RNAi-mediated silencing of GFP. Cells stably expressing EGFPwere cotransfected with the designated oligo and a plasmid thatexpresses a scrambled shRNA (neg), shRNA switch S1, or an shRNAtargeting a different region of the EGFP mRNA (sh′). Mean fluorescencerelative to negative based on flow cytometry measurements of transfectedcells. The guide strands are “S1” and “sh′”; the 2′-O-Methyl nucleotidesare “2′OMe anti” and “2′OMe rev”; and all the nucleotides except for thethree 3′-end nucleotides in “deoxy anti” are deoxynucleotides. Errorbars represent one standard deviation from duplicate transfected wells.

FIG. 9 shows functionality of shRNA switches in different cell lines.Cells lacking endogenous EGFP expression were cotransfected with theshRNA construct and an EGFP expression plasmid in the presence (▪) orabsence (□) of 3 mM theophylline. Mean fluorescence values werenormalized to red fluorescence values (DsRed-Express) contributed by theshRNA construct. These values were then normalized to those of cellstransfected with a scrambled shRNA (neg). Error bars represent onestandard deviation from triplicate transfected wells.

FIG. 10 shows the derived model equation and model fit of thetheophylline response curve for S1 data. The value of f_(shRNA) was setby a separate transfection experiment with the original shRNA targetingEGFP (sh) under the same conditions. The value of e was determined fromthe average basal expression levels of shRNA switches that highly favorthe active conformation (S5, S7, S9, S1). Parameters K_(Comp), K_(Apt)and h were produced by a least-squares fit (−) to the S1 data ().Parameter values are reported to the right of the plot.

FIG. 11 depicts methods to calculate ΔG from shRNA switch sequenceinformation. Base-pairing probabilities of base-pairs designated byarrows were used for the PF method. The boxed sections designate themajor stem in the active and inactive conformations used under theSstems method.

FIG. 12 shows the use of alternative methods for relating shRNA switchsequence and in vivo basal expression levels. ΔG was calculated(ΔG_(method)) for shRNA switches S1-10 using RNA secondary structureprediction algorithms. Plots relating ΔG_(method) and measured basalexpression level for shRNA switches S1-10, where ΔG was calculated usingthe MFE method or the PF method. A three-parameter equation with thesame mathematical form as the model was fit by least-squares analysis toeach data set.

FIG. 13 shows theophylline response curves for the initial (S1, ) andoptimized (S13, ) shRNA switches. Median fluorescence values from flowcytometry analysis were normalized to that of untransfected cells in thesame well. Error bars represent one standard deviation from duplicatetransfected wells.

FIG. 14 shows theophylline-mediated regulation of endogenous La proteinwith shRNA switches. (A) qRT-PCR of La protein mRNA from HEK293TtTA-d2EGFP cells transfected in the presence (▪) or absence (□) of 1.5mM theophylline. Calculated free energy differences from the stems,minimal free energy (MFE) and partition function (PF) method aredisplayed below each shRNA switch. Error bars represent quadruplicateqRT-PCR measurements. (B) Curve fit to extrapolate empirical parametersC1-3. Dashed line marks knockdown achieved by base shRNA. (C) Modelpredictions for dynamic range using empirical parameter values for theGFP experiments along with f_(shRNA)=0.6 and e=0.72 (gray curve) andextrapolated values from the La experiments (black curve). (D)Relationship between dynamic range (η) and the free energy difference.The dashed line represents the apparent increase in La mRNA levels upontheophylline addition observed for the base shRNA (shL).

FIG. 15 shows representative histograms (top row) and dot plots (bottomrow) for HEK293T tTA-d2EGFP cells transiently transfected with a plasmidexpressing DsRed-Express and either a scrambled shRNA (Scr), the baseshRNA targeting EGFP (sh), or shRNA switch S13. Gates in the histogramcapture the transfected (H) and untransfected (L) populations in eachwell. The calculation of GFP(%) used in the main text is shown on theright. Median GFP levels of each transfected population were normalizedto that of untransfected cells in the same well, thereby reducingwell-to-well variability.

DETAILED DESCRIPTION OF THE INVENTION 1. Overview

The present invention provides a universal and extensiblepolynucleotide-based platform for engineering ligand-controlled generegulatory systems that exhibit tunable regulation of gene expression,design modularity, and target specificity. The invention also providespolynucleotides engineered based on these design principles.

Specifically, the application describes a framework for the reliable denovo construction of modular, portable, and scalable control systemsthat can be used to achieve flexible regulatory properties, such as up-and down-regulation of target expression levels and tuning of regulatoryresponse to fit application-specific performance requirements, therebyexpanding the utility of our platforms to a broader range ofapplications. For example, these switch platforms may be applied to theconstruction of transgenic regulatory control systems that areresponsive to cell permeable, exogenous molecules of interest for agiven network. In regulating sets of functional proteins, these switchescan act to rewire information flow through cellular networks andreprogram cellular behavior in response to changes in the cellularenvironment. In regulating reporter proteins, the subject regulatednucleic acids (or switches) can serve as synthetic cellular sensors tomonitor temporal and spatial fluctuations in the levels of diverse inputmolecules. The switch platforms described here represent powerful toolsfor constructing ligand-controlled gene regulatory systems tailored torespond to specific effector molecules and enable regulation of targetgenes in various living systems. The design principles described hereinenable the de novo design of such versatile switches. Due to theirgeneral applicability, our platforms offer broad utility forapplications in synthetic biology, biotechnology, and health andmedicine.

Thus one aspect of the invention relates to a sensor domain-regulated(e.g., aptamer-regulated) nucleic acid and methods and compositionscomprising these nucleic acids for modulating (e.g., attenuating) geneexpression in a cell. Another aspect relates to sensor domain-regulated(e.g., aptamer-regulated) nucleic acids that may be employed as in vivosensors to detect the presence, absence, or amount of a molecule in asample. For example, such nucleic acids may be used to sense changes inan intracellular condition, such as changes in ligand concentration, pH,temperature, etc. After sensing the change, an output signal from areporter gene (such as expression of a fluorescent protein, or activityof an enzyme) may be used to monitor the changes. In certainembodiments, the sensor domain-regulated (e.g., aptamer-regulated)nucleic acid may alter the detected change in the condition through thecoupled actuator domain, thus providing a feedback regulation.

The sensor domain-regulated (e.g., aptamer-regulated) nucleic acids maybe either trans-acting or cis-acting. By trans-acting, it is meant thatthe switches of the present inventions exert their ligand-dependentactivity on a molecule, e.g. another nucleic acid, that is differentfrom the switch, e.g. not linker through a phosophodiester (orequivalent) backbone linker, and even more preferably not covalentlylinked to the switch at all. By cis-acting, it is meant that theswitches of the present inventions exert their ligand-dependent activityon itself, or an otherwise heterologous molecule that is covalentlylinked to the switch, e.g., through a phosophodiester (or equivalent)backbone linker.

Thus, one aspect of the present invention provides engineered, sensordomain (aptamer) regulated nucleic acids that are powerful, allostericregulators of gene expression. A general design of such nucleic acids isbased on conformational dynamics of nucleic acid folding to create amodular molecule comprising a modular actuator domain, a modular sensor(aptamer) domain, and an information transmission domain (ITD) thatfunctionally couples the actuator domain and the sensor (aptamer) domainsuch that the latter two domains can remain truly modular (seedefinition below). In contrast to the strand-slippage mechanism (seedefinition below) based information transmission domain, the ITD of thesubject invention is based on the strand-displacement mechanism (seedefinition below) that is amenable to rational design. Such astrand-displacement mechanism uses competitive binding of two nucleicacid sequences (e.g., the competing strand and the switching strand) toa general transmission region of the switch (e.g., the base stem of theaptamer) to result in disruption or restoration of the actuator domainin response to restoration of the sensor domain.

A further feature of the subject information transmission domain is thatthe switching strand and the competing strand of the ITD are in acontinuous strand of sequence (e.g., the switching strand may be withina loop structure in at least one conformation of the sensordomain-regulated nucleic acid, and does not have a free 5′-OH or 3′-OHgroup). This design feature eliminates any free-floating ends of theswitching strand and/or the competing strand and localizes thecompetitive hybridization event to two strands that arecontinguous/linked, thereby improving the kinetics ofstrand-displacement.

The subject sensor domain-regulated nucleic acids are preferablydesigned such that it can adopt at least two distinct conformations. Inone conformation, the sensor domain is capable of binding to a ligand,and the actuator domain may resume one activity state (e.g., more activestate or less active state). In the other conformation, the sensordomain is incapable of binding to the ligand, and the actuator domainmay resume another activity state. The conformation change of the sensordomain may be transmitted through the information transmission domain tothe coupled actuator domain, so that the actuator domain adopts one ofthe two activity states depending on whether the sensor domain can orcannot bind the ligand.

While not wishing to be bound by any particular theory, the moleculedistributes between two conformations, partly based on the free energydifferences between the two conformations. Ligand binding to one of thetwo conformations provides additional stability to the complex, thusshifting the distribution towards the conformation capable ofligand-binding, and thus favoring the active state of the actuatordomain associated with the ligand-binding sensor domain. Thus at themacro level, it appears that ligand-binding has caused a conformationchange from one conformation to the other, and “conformation change” maybe used herein to describe the ligand binding-induced shift inpartitioning between the two conformations.

The subject regulated nucleic acid platform is flexible, enabling bothpositive and negative regulation as a function of ligand concentration.For example, the ligand-bound sensor domain may be associated with themore active form of the actuator domain, or with the less active form ofthe actuator domain.

The switching dynamics of aptamer-regulated nucleic acids are amenableto tuning by forward engineering design strategies based onthermodynamic properties of nucleic acids. Altering the free energy ofthe actuator domain and/or the sensor domain alters the conformationaldynamics of these molecules in a predictable fashion. Specifically,decreasing the stability of the actuator domain associated with oneactivity state (by, for example, reducing the length of perfectlymatched base pairs in a stem structure within the actuator domain, orlower the quality of base-pairing) may favor a shift in partitioningbetween the two conformations to the other activity state of theactuator domain (and its associated sensor domain conformation), thusincreasing or decreasing the ligand concentration necessary to inducethe equilibrium shift between the two conformations. This may alsochange the dynamic range of the switch in addition to ligandconcentration dependence). On the other hand, changing the size of thesensor domain does not necessarily alter the conformational changebetween the two conformations, but may change the efficiency with whichthe switch is processed (by RNase III, for example, when the actuatordomain is a Dicer substrate). Likewise, changing the affinity of thesensor (aptamer) domain for the ligand provides yet another fine tuningstrategy for the subject sensor-regulated polynucleotides. These finetuning strategies may be used in combination to provide flexible yetpredictable changes in activity of the subject sensor domain-regulatednucleic acids in response to different ligand concentrations orenvironmental condition changes.

In addition, the aptamer-regulated nucleic acid platform is fullymodular, enabling ligand response and actuator function (e.g.,transcript targeting) to be engineered by swapping domains within thesubject regulated nucleic acid. This provides a platform for theconstruction of tailor-made sensor domain regulated nucleic acids for avariety of different ligands. Ligand binding of the aptamer domain inaptamer-regulated nucleic acids is designed separately from thetargeting capability of the actuator domain by swapping only the aptamerdomain. Likewise, the targeting capability of the actuator domain can bedesigned separately from the ligand binding of the aptamer domain byswapping the effector domain so that a different gene or molecule istargeted without affecting the aptamer domain. This feature is madepossible by the unique property of the subject information transmissiondomain that employs the strand displacement mechanism. Thus, the subjectsensor domain-regulated nucleic acids present a powerful, flexiblemethod of tailoring spatial and temporal gene expression in both naturaland engineered contexts.

The subject sensor domain-regulated nucleic acids are novel, allostericregulators of gene expression that can potentially function across adiverse range of organisms, from prokaryotes to humans, making themextremely useful in many different applications. For example, thesubject regulated nucleic acids presents a powerful tool for genetherapy applications, where one would like to target specifictranscripts in response to specific cellular environments that areindicative of a diseased state (Watkins et al., Curr Opin Mol Ther 4,224-8 (2002)). As emerging technologies enable the metabolic profilingof disease states (Koch, J Biol Chem 219, 181-8 (1956)),aptamer-regulated nucleic acids can be designed to respond to variousmetabolic markers, or a specific concentration range of a metabolicmarker, partly owning to the susceptibility of the subject sensordomain-regulated nucleic acids to the various fine-tuning strategies.For instance, aptamer-regulated nucleic acids can be constructed toinhibit genes necessary for cell growth and division in response tooncogenic proteins or isoforms. One can also anticipate an exogenouslydelivered aptamer-regulated nucleic acid comprising an actuator domainthat is an antisense construct or RNase III substrate acting as atherapeutic molecule, similar to exogenously delivered antisenseoligonucleotides or RNAi therapeutic agents, thereby extending thefunctionality of current antisense/RNAi therapies by introducingligand-specific or cell type-specific action to an already highlytargeted therapy.

The subject regulated nucleic acids can further be used to engineernovel regulatory pathways and control loops for applications inmetabolic engineering (Khosla et al., Nat Rev Drug Discov 2, 1019-25(2003)) and synthetic circuit design (Kobayashi et al., Proc Natl AcadSci USA 101, 8414-9 (2004)) by enabling the cell to sense and respond tointracellular metabolite levels and environmental signals. Because theregulated nucleic acids activity is tunable over a range of ligandconcentrations, switches can be designed to inhibit or activate genesonly when certain metabolites exceed or go below certain concentrations.Balancing heterologous gene expression in biosynthetic pathways (Berenset al., Bioorg Med Chem 9, 2549-56 (2001)) to maximize product yield canbe achieved with aptamer-regulated nucleic acids that regulateexpression of biosynthetic genes in response to pathway intermediatelevels. Synthetic gene circuits have recently been used to understandand model cellular networks (Nagai et al., Nat Biotechnol 20, 87-90(2002)) and to achieve cellular control as a step towards “programmable”cell behavior (Watkins et al., Curr Opin Mol Ther 4, 224-8 (2002)). Genecircuits can be built using combinations of aptamer-regulated nucleicacids as regulators for precise control schemes. Aptamer-regulatednucleic acids will be useful tools in building and characterizingcircuits that accurately model natural regulatory pathways and yieldfurther insight into these prevalent regulation schemes.

Finally, sensor domain-regulated nucleic acids present new tools forcellular imaging, measuring, and detection strategies enablingprogrammable concentration-specific detection of intracellularmolecules. Such nucleic acids offer a unique platform to createtailor-made cellular sensors and “smart” regulators that potentially cantarget any gene in response to any target ligand, creating new avenuesfor cellular control and engineering.

2. Definitions

“Actuator domain” refers to a switch domain that encodes the systemcontrol function. In certain embodiments, the actuator domain encodesthe gene-regulatory function, and the actuator domain is not a ribozyme,such as a hammerhead ribozyme.

As used herein, a “bulge” is a sequence of nucleotides that is notpaired with another strand and is flanked on both sides bydouble-stranded nucleic acid sequences. In certain embodiments, a bulgeis located within a stem. When a bulge is located within a stem, thenucleotides of the bulge are considered to be part of the stem. Incertain embodiments, a stem may comprise more than one bulge. In certainembodiments, one or both strands of the stem contain a bulge.

“Communication module” refers to a sequence element that typically formsan imperfectly paired double-stranded stem that can adopt different basepairs between nucleotides through a “slip-structure” mechanism. Acommunication module may be a type of information transmission domainthat transmits the binding state of the aptamer domain to the adjacentactuator domain through a helix-slipping mechanism. A communicationmodule does not generally act in a modular fashion with other switchdomains.

“Competing strand” refers to the nucleic acid sequence within astrand-displacement domain that is bound to the general transmissionregion of the switch when the sensor domain is in one conformation, suchas the restored conformation (i.e., in the presence of ligand in thishypothetical situation). The competing strand competes for binding withthe switching strand, which is initially bound to this transmissionregion (for example, in the absence of ligand).

“Complementary” refers to a nucleotide or nucleotide sequence thathybridizes to a given nucleotide or nucleotide sequence. For instance,for DNA, the nucleotide A is complementary to T, and vice versa, and thenucleotide C is complementary to G, and vice versa. For instance, inRNA, the nucleotide A is complementary to the nucleotide U, and viceversa, and the nucleotide C is complementary to the nucleotide G, andvice versa. Complementary nucleotides include those that undergo Watsonand Crick base pairing and those that base pair in alternative modes.For instance, as used herein for RNA, the nucleotide G is complementaryto the nucleotide U and vice versa, and the nucleotide A iscomplementary to the nucleotide G and vice versa. Therefore, in an RNAmolecule, the complementary base pairs are A and U, G and C, G and U,and A and G. Other combinations, e.g., A and C, A and A, G and G, or Cand U, are considered to be non-complementary base pairs.

Due to the binding energy differences between different base pairs, the“quality of complementarity” also varies, and may be explored to finetune the free energy differences between different conformations of thesubject regulated polynucleotides. For example, the G-C base pairexhibits the highest binding affinity, and thus is said to have a higherquality of binding than that of an A-T or A-U pair, or a G-U pair, etc.Depending on specific needs, a Watson-Crick base pair may be replace byanother (stronger or weaker) Watson-Crick base pair, or a wobble basepair to alter the quality of complementarity of any region in thesubject regulated nucleic acid.

A “complementary sequence” comprises individual nucleotides that arecomplementary to the individual nucleotides of a given sequence, wherethe complementary nucleotides are ordered such that they will pairsequentially with the nucleotides of the given sequence. Such acomplementary sequence is said to be the “complement” of the givensequence. For example, complements of the given sequence, 5′-ACUAGUC-3′,include 3′-UGAUCAG-5′ and 3′-UGGACGG-3′, among others. In the lattersequence, the third and sixth base pairs are both non-Watson and CrickG/U complementary base pairs.

“Component” is a part of a system that encodes a distinct activity orfunction.

“Composability” refers to a property of a system that indicates itsability to be comprised of components that can be selected and assembledin a modular fashion to achieve a desired system performance. Forexample, in certain embodiments, composability refers to the ability ofthe individual domains of the control system to be modularly linkedwithout disrupting their activities.

“Do/does not bind” as used herein to describe aptamer-ligand binding,does not mean that there is absolutely no binding at all. Compared to anaptamer that does bind the ligand (a “binding aptamer”), the K_(Apt)(association constant for binding between ligand and aptamer) for theaptamer that “does not bind” the ligand is at least about 10-fold,100-fold, 1000-fold or more larger than that of the binding aptamer, andthus its binding affinity for the ligand is at least about 10-fold,100-fold, 1000-fold or more weaker than that of the binding aptamer.

“Engineering design principle” refers to a required property of aconstructed system that enables use by others.

“Framework” refers to a basic conceptual structure that is used to solvea complex product design issue. As used here, the framework is used toreliably design and construct specific instances of RNA switches. Theconceptual structure of the subject framework comprises specifiedengineering design principles and design strategies that enableextensible and reusable system design.

“Helix-slipping domain” refers to a subset of information transmissiondomains that act through a helix-slipping mechanism. The helix-slippingdomain is also referred to as the communication module.

“Helix-slipping mechanism” refers to an information transmissionmechanism that is based on an information transmission domain thatfunctions through a helix-slipping event and does not allow for rationaldesign. Such a helix-slipping event uses a communication module (orhelix-slipping domain) within the general transmission region of theswitch (e.g., the base stem of the aptamer) to result in disruption orrestoration of the actuator domain in response to restoration of thesensor domain.

“Information transmission domain” refers to a switch domain that encodesthe function of transmitting information between the sensor domain andthe actuator domain.

“Information transmission mechanism” refers to a general mechanism fortransmitting information between the sensor domain and the actuatordomain of a switch. In certain embodiments, this mechanism regulates theactivity of the actuator domain in response to the binding state of thesensor domain.

“Loop” refers to a sequence of nucleotides that is not paired withanother strand. In certain embodiments, a loop is between 1 to 20nucleotides long, 2-10 nucleotides long, or 3-8 nucleotides long.

“Modular” refers to a property of a system composed of modules thatindicates whether the modules can by interchanged as parts withoutchanging the interface between modules or the modules themselves.

“Module” refers to a self-contained system component that has a welldefined interface with other system components.

“Nucleotide” refers to naturally- and non-naturally-occurringnucleotides and nucleotide analogs. Nucleotides include, but are notlimited to, adenosine, cytosine, guanosine, thymidine, uracil,4-acetylcytosine, 8-hydroxy-N-6-methyladenosine, aziridinyl-cytosine,pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil,5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil,5-carboxy-methylaminomethyluracil, dihydrouracil, inosine,N6-iso-pentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonyl-methyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine and2,6-diaminopurine.

“Nucleic acid,” “nucleic acid sequence,” “nucleic acid molecule,” and“polynucleotide” refer to a DNA sequence or analog thereof, or an RNAsequence or analog thereof. Nucleic acids are formed from nucleotides,including, but not limited to, the nucleotides listed above.

“Platform” refers to a general framework on which specific applicationscan be implemented. In certain embodiments, the platform enablesspecific instances of switches to be built in a standardized manner.

“Portability” refers to a property of a system that indicates itsability to be implemented in environments different from that in whichit was originally designed. In certain embodiments, portability refersto the ability of the control system to be implemented in differentorganisms.

“Reliability” refers to a property of a system that indicates itsability to perform and maintain its functions under a set of specifiedconditions. In certain embodiments, reliability refers to the ability ofthe information transmission domain to standardize the transmission ofinformation between the sensor and actuator domains.

“Scalability” refers to a property of a system that indicates itsability to handle increasing work. In certain embodiments, scalabilityrefers to the ability of the control system to be implemented acrossbroad application space by being able to forward design its response todifferent molecular information.

A “stem” is a double-stranded nucleic acid motif formed by inter- orintra-molecular base pairing, which may or may not include mismatchedbase pairs or “bulges.” In certain embodiments, a stem comprises 2 toabout 40, or 2 to about 20 complementary base pairs. In certainembodiments, a stem comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, or 21 complementary base pairs.

In certain embodiments, at least 30% of the nucleotides in a stem arepart of a complementary base pair. The remaining base pairs may bemismatched, non-complementary base pairs, or may be part of a bulge. Incertain embodiments, at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or100% of the nucleotides in the stem are part of a complementary basepair.

“Switch” refers to a molecule that can adopt at least two differentconformational states, where each state is associated with a differentactivity of the molecule. Often a ligand can bind to one or moreconformations of the switch, such that the presence of the ligand shiftsthe distribution or equilibrium across the adoptable conformations andtherefore regulates the activity of the switch molecule. In certainembodiments, switch refers to an RNA molecule that can adopt differentstructures that correspond to different gene regulatory activities. AnRNA switch is an exemplary embodiment of the subject ligand-controlledgene-regulatory system.

“Switch domain” refers to a component of a switch that encodes adistinct activity or function.

“Switching strand” refers to the nucleic acid sequence within a stranddisplacement domain that is bound to the general transmission region ofthe switch when the sensor domain is, for example, in the disruptedconformation (i.e., in the absence of ligand in this hypotheticalsituation). The switching strand is displaced by the competing strandin, for example, the presence of ligand (in this hypotheticalsituation).

“Sensor domain” refers to a switch domain that encodes a ligand-bindingfunction. In certain embodiments, the sensor domain comprises an RNAaptamer sequence.

“Strand-displacement domain” refers to a subset of informationtransmission domains that act through a strand-displacement mechanism.

“Strand-displacement mechanism” refers to an information transmissionmechanism that is based on the rational design of an informationtransmission domain that functions through a strand-displacement event.Such a strand-displacement event uses competitive binding of two nucleicacid sequences (e.g., the competing strand and the switching strand) toa general transmission region of the switch (e.g., the base stem of theaptamer) to result in disruption or restoration of the actuator domainin response to restoration of the sensor domain.

“Universal” refers to a system property that indicates its ability tomaintain function across different applications, environments, andcomponent interfaces. In certain embodiments, a universal system iscomposed of the five engineering design principles (scalability,portability, utility, composability, and reliability) and results in thespecified extensible platform for RNA switch construction.

“Utility” refers to a property of a system that indicates its ability tobe of practical use. In certain embodiments, utility refers to theability of the control system to interface with different functionallevel components to enable forward design of the function that is beingcontrolled by the system.

Other terms used herein and in the claims adopt their plain meanings aswould have been understood by one of skill in the relevant art, that arenot inconsistent with the usages in the instant specification.

3. Sensor-Regulated Polynucleotides

The sensor-regulated polynucleotides of the invention comprise a modularactuator domain, a modular sensor domain, and an informationtransmission domain. Such polynucleotides may comprise DNA or RNA, or acombination thereof. The polynucleotides may also be single-stranded ordouble-stranded. The single-stranded polynucleotide may comprise one ormore double-stranded regions (or stems) due to intramolecularinteraction (e.g., RNA secondary structure). If one or morephosophodiester linkage between the nucleotides are broken, the foldedpolynucleotide may in fact be double-stranded while maintainingsubstantially the same secondary structure.

The sensor-regulated polynucleotides may further comprise a functionalgroup or a functional agent, e.g., an intercalator or an alkylatingagent. The sensor-regulated polynucleotides may comprise synthetic ornon-natural nucleotides and analogs (e.g., 6-mercaptopurine,5-fluorouracil, 5-iodo-2′-deoxyuridine and 6-thioguanine) or may includemodified nucleic acids. Exemplary modifications include cytosineexocyclic amines, substitution of 5-bromo-uracil, backbonemodifications, methylations, and unusual base-pairing combinations.Additional analogs include at least one modified base moiety which isselected from the group including but not limited to 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxytriethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil; beta-D-mannosylqueosine,5-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

The sensor-regulated polynucleotides may also include labels, such asfluorescent, radioactive, chemical, or enzymatic labels.

In certain preferred embodiments, the modular sensor domain comprises anaptamer that responds to ligand binding to favors an allosteric changein the modular actuator domain, and alters the ability of the actuatordomain to interact with its target molecule. Ligand binding, therefore,switches the actuator domain from “off” to “on,” or vice versa. Thesensor-regulated polynucleotides, therefore, act as a switch whoseactivity is turned “off” and “on” in response to ligand binding.

The response of the sensor (aptamer) domain to the ligand may alsodepend on the ligand identity and/or the amount or concentration ofligand exposed to the sensor (aptamer) domain. For example, an aptamermay bind small molecules, such as drugs, metabolites, intermediates,cofactors, transition state analogs, ions, metals, nucleic acids, andtoxins. Alternatively, an aptamer may bind natural and syntheticpolymers, including proteins, peptides, nucleic acids, polysaccharides,glycoproteins, hormones, receptors and cell surfaces such as cell wallsand cell membranes.

In certain other embodiments, the sensor domain of the subject ligandcontrolled, sensor-regulated polynucleotide is responsive toenvironmental changes. Environmental changes include, but are notlimited to changes in pH, temperature, osmolarity, or saltconcentration.

Aptamers

An “aptamer” may be a nucleic acid molecule, such as RNA or DNA that iscapable of binding to a specific molecule with high affinity andspecificity (Ellington et al., Nature 346, 818-22 (1990); and Tuerk etal., Science 249, 505-10 (1990)). Exemplary ligands that bind to anaptamer include, without limitation, small molecules, such as drugs,metabolites, intermediates, cofactors, transition state analogs, ions,metals, nucleic acids, and toxins. Aptamers may also bind natural andsynthetic polymers, including proteins, peptides, nucleic acids,polysaccharides, glycoproteins, hormones, receptors and cell surfacessuch as cell walls and cell membranes. The binding of a ligand to anaptamer, which is typically RNA, causes or favors a conformationalchange in the actuator domain and alters its ability to interact withits target molecule. Therefore, ligand binding affects the actuatordomain's ability to mediate gene inactivation, transcription,translation, or otherwise interfere with the normal activity of thetarget gene or mRNA, for example.

An aptamer will most typically have been obtained by in vitro selectionfor binding of a target molecule. However, in vivo selection of anaptamer is also possible. Aptamers have specific binding regions whichare capable of forming complexes with an intended target molecule in anenvironment wherein other substances in the same environment are notcomplexed to the nucleic acid. The specificity of the binding is definedin terms of the comparative dissociation constants (K_(D)) of theaptamer for its ligand as compared to the dissociation constant of theaptamer for other materials in the environment or unrelated molecules ingeneral. A ligand is one which binds to the aptamer with greateraffinity than to unrelated material. Typically, the K_(D) for theaptamer with respect to its ligand will be at least about 10-fold lessthan the K_(D) for the aptamer with unrelated material or accompanyingmaterial in the environment. Even more preferably, the K_(D) will be atleast about 50-fold less, more preferably at least about 100-fold less,and most preferably at least about 200-fold less. An aptamer willtypically be between about 10 and about 300 nucleotides in length. Morecommonly, an aptamer will be between about 30 and about 100 nucleotidesin length.

The terms “nucleic acid molecule” and “polynucleotide” refer todeoxyribonucleotides or ribonucleotides and polymers thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogues of naturalnucleotides which have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions) andcomplementary sequences and as well as the sequence explicitlyindicated. Specifically, degenerate codon substitutions may be achievedby generating sequences in which the third position of one or moreselected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini etal., Mol. Cell. Probes 8:91-98 (1994)). Also included are moleculeshaving naturally occurring phosphodiester linkages as well as thosehaving non-naturally occurring linkages, e.g., for stabilizationpurposes. The nucleic acid may be in any physical form, e.g., linear,circular, or supercoiled. The term nucleic acid is used interchangeablywith oligonucleotide, gene, cDNA, and mRNA encoded by a gene.

Aptamers are readily made that bind to a wide variety of molecules. Eachof these molecules can be used as a modulator of gene expression usingthe methods of the invention. For example, organic molecules,nucleotides, amino acids, polypeptides, target features on cellsurfaces, ions, metals, salts, saccharides, have all been shown to besuitable for isolating aptamers that can specifically bind to therespective ligand. For instance, organic dyes such as Hoechst 33258 havebeen successfully used as target ligands for in vitro aptamer selections(Werstuck and Green, Science 282:296-298 (1998)). Other small organicmolecules like dopamine, theophylline, sulforhodamine B, and cellobiosehave also been used as ligands in the isolation of aptamers. Aptamershave also been isolated for antibiotics such as kanamycin A,lividomycin, tobramycin, neomycin B, viomycin, chloramphenicol andstreptomycin. For a review of aptamers that recognize small molecules,see Famulok, Science 9:324-9 (1999).

In certain embodiments, the ligand of the aptamer of anaptamer-regulated nucleic acid of the invention is a cell-permeable,small organic molecule. Small organic molecules which do not have ageneral inhibitory effect on translation are preferred as ligands. Thesmall molecule preferably also exhibits in vivo persistence sufficientfor achieving the desired level of inhibition of translation. Themolecules also can be screened to identify those that are bioavailableafter, for example, oral administration. In certain embodiments of theinvention, the ligand is nontoxic. The ligand may optionally be a drug,including, for example, a steroid. However, in some of the methods ofcontrolling gene expression, it is preferable that the ligand bepharmacologically inert. In some embodiments, the ligand is apolypeptide whose presence in the cell is indicative of a disease orpathological condition. In other embodiments, the ligand for an aptameris an antibiotic, such as chloramphenicol. In an alternative embodiment,the ligand of the aptamer is an organic dye such as Hoeschst dye 33258.In still another embodiment, the ligand may be a metal ion. In aspecific embodiment, the aptamer domain of an aptamer-regulated nucleicacid responds to binding to caffeine.

The aptamer-regulated nucleic acid of the invention can be composedentirely of RNA. In other embodiments of the invention, however, theaptamer-regulated nucleic acid can instead be composed entirely of DNA,or partially of DNA, or partially of other nucleotide analogs. Tospecifically inhibit translation in vivo, aptamer-regulated RNAs arepreferred. Such aptamer-regulated RNAs are preferably introduced into acell as a DNA that encodes the aptamer-regulated nucleic acid sequencesuch that transcription results in the aptamer-regulated RNA.Alternatively, an aptamer-regulated RNA itself can be introduced into acell.

Aptamers are typically developed to bind particular ligands by employingknown in vivo or in vitro (most typically, in vitro) selectiontechniques known as SELEX (Ellington et al., Nature 346, 818-22 (1990);and Tuerk et al., Science 249, 505-10 (1990)). Methods of makingaptamers are also described in, for example, U.S. Pat. No. 5,582,981,PCT Publication No. WO 00/20040, U.S. Pat. No. 5,270,163, Lorsch andSzostak, Biochemistry, 33:973 (1994), Mannironi et al., Biochemistry36:9726 (1997), Blind, Proc. Nat'l. Acad. Sci. USA 96:3606-3610 (1999),Huizenga and Szostak, Biochemistry, 34:656-665 (1995), PCT PublicationNos. WO 99/54506, WO 99/27133, WO 97/42317 and U.S. Pat. No. 5,756,291.

Generally, in their most basic form, in vitro selection techniques foridentifying aptamers involve first preparing a large pool of DNAmolecules of the desired length that contain at least some region thatis randomized or mutagenized. For instance, a common oligonucleotidepool for aptamer selection might contain a region of 20-100 randomizednucleotides flanked on both ends by an about 15-25 nucleotide longregion of defined sequence useful for the binding of PCR primers. Theoligonucleotide pool is amplified using standard PCR techniques,although any means that will allow faithful, efficient amplification ofselected nucleic acid sequences can be employed. The DNA pool is then invitro transcribed to produce RNA transcripts. The RNA transcripts maythen be subjected to affinity chromatography, although any protocolwhich will allow selection of nucleic acids based on their ability tobind specifically to another molecule (e.g., a protein or any targetmolecule) may be used. In the case of affinity chromatography, thetranscripts are most typically passed through a column or contacted withmagnetic beads or the like on which the target ligand has beenimmobilized. RNA molecules in the pool which bind to the ligand areretained on the column or bead, while nonbinding sequences are washedaway. The RNA molecules which bind the ligand are then reversetranscribed and amplified again by PCR (usually after elution). Theselected pool sequences are then put through another round of the sametype of selection. Typically, the pool sequences are put through a totalof about three to ten iterative rounds of the selection procedure. ThecDNA is then amplified, cloned, and sequenced using standard proceduresto identify the sequence of the RNA molecules which are capable ofacting as aptamers for the target ligand. Once an aptamer sequence hasbeen successfully identified, the aptamer may be further optimized byperforming additional rounds of selection starting from a pool ofoligonucleotides comprising the mutagenized aptamer sequence. For use inthe present invention, the aptamer is preferably selected for ligandbinding in the presence of salt concentrations and temperatures whichmimic normal physiological conditions.

An improved aptamer selection scheme is described in the co-owned andco-pending U.S. application Ser. No. 12/218,628, filed on Jul. 16, 2008,the entire content of which is incorporated herein by reference.

One can generally choose a suitable ligand without reference to whetheran aptamer is yet available. In most cases, an aptamer can be obtainedwhich binds the ligand of choice by someone of ordinary skill in theart. The unique nature of the in vitro selection process allows for theisolation of a suitable aptamer that binds a desired ligand despite acomplete dearth of prior knowledge as to what type of structure mightbind the desired ligand.

For an aptamer to be suitable for use in the present invention, thebinding affinity of the aptamer for the ligand must be sufficientlystrong and the structure formed by the aptamer when bound to its ligandmust be significant enough so as to switch an aptamer-regulated nucleicacid of the invention between “on” and “off” states of anaptamer-regulated nucleic acid.

The association constant for the aptamer and associated ligand ispreferably such that the ligand functions to bind to the aptamer andhave the desired effect at the concentration of ligand obtained uponadministration of the ligand. For in vivo use, for example, theassociation constant should be such that binding occurs well below theconcentration of ligand that can be achieved in the serum or othertissue, preferably well below the concentration of ligand that can beachieved intracellularly since cellular membranes may not besufficiently permeable to allow the intracellular ligand concentrationto approach the level in the serum or extracellular environment.Preferably, the required ligand concentration for in vivo use is alsobelow that which could have undesired effects on the organism.

Actuator/Effector Domain

An actuator/effector nucleic acid domain may comprise an antisensenucleic acid or a DNA. An effector nucleic acid domain may also comprisea sequence that can be used as an RNAi sequence or precursor that givesrise to siRNA or miRNA. Yet other actuator domains can be an shRNA orprecursor thereof, an RNase III substrate, an alternative splicingelement, or an RNAi targeting sequence. In certain embodiments, theactuator of the invention does not include ribozymes or other catalyticnucleic acids.

In preferred embodiments, ligand binding at the sensor (aptamer) domainmediates a change in the conformational dynamics of these molecules thatallows or prevents the actuator domain to interact with a target nucleicacid, for example, an mRNA.

In one embodiment, the actuator domain of an aptamer-regulated nucleicacid interacts with a target gene by nucleic acid hybridization. Forinstance, an aptamer-regulated nucleic acid may comprise an actuatordomain that comprises a hybridization sequence that hybridizes to atarget sequence of a gene and an aptamer domain that binds to a ligand.The binding of the ligand to the aptamer domain favors a conformationalchange in the aptamer-regulated nucleic acid that alters the ability(such as availability and/or T_(m)) of the hybridization sequence of theactuator domain to hybridize to a target sequence. Furthermore, anactuator domain may modulate the expression or activity of its target byany method known in the art. In one embodiment, the actuator domain ofan aptamer-regulated nucleic acid comprises an antisense sequence andacts through an antisense mechanism in modulating expression of a targetgene. For instance, an aptamer-regulated nucleic acid may comprise anactuator domain that comprises an antisense sequence for inhibitingexpression of a target gene and an aptamer domain that binds to aligand. The binding of the ligand to the aptamer domain causes aconformational change in the aptamer-regulated nucleic acid that altersthe ability of the antisense sequence of the actuator domain to inhibitexpression of the target sequence.

In another embodiment, the actuator domain of an aptamer-regulatednucleic acid comprises an actuator domain that comprises an RNAisequence and acts through an RNAi or miRNA mechanism in modulatingexpression of a target gene. For instance, an aptamer-regulated nucleicacid may comprise an actuator domain that comprises a miRNA or siRNAsequence for inhibiting expression of a target gene and an aptamerdomain that binds to a ligand. The binding of the ligand to the aptamerdomain causes a conformational change in the aptamer-regulated nucleicacid that alters the ability of the miRNA or siRNA sequence of theactuator domain to inhibit expression of the target sequence. In oneembodiment, an effector domain comprises a miRNA or siRNA sequence thatis between about 19 nucleotides and about 35 nucleotides in length, orpreferably between about 25 nucleotides and about 35 nucleotides. Incertain embodiments, the actuator domain is a hairpin loop that may beprocessed by RNase III enzymes (e.g., Drosha and Dicer). As used herein,the term “RNAi” means an RNA-mediated mechanism for attenuating geneexpression and includes small RNA-mediated silencing mechanisms.RNA-mediated silencing mechanisms include inhibition of mRNA translationand directed cleavage of targeted mRNAs. Recent evidence has suggestedthat certain RNAi constructs may also act through chromosomal silencing,i.e., at the genomic level, rather than, or in addition to, the mRNAlevel. Thus, the sequence targeted by the actuator domain can also beselected from untranscribed sequences that regulate transcription of atarget gene at the genomic level.

The methods described herein may employ an expression vector having acoding sequence that is transcribed to produce one or moretranscriptional products that produce an aptamer-regulated nucleic acidin the treated cells. Expression vectors appropriate for producing anaptamer-regulated nucleic acid are well-known in the art. For example,the expression vector is selected from an episomal expression vector, anintegrative expression vector, and a viral expression vector. In anotherpreferred embodiment, the aptamer-regulated nucleic acid comprises ahairpin RNA which is processed to an siRNA in the treated cells.

In certain embodiments, the expression construct can be designed toinclude one or more subject regulated polynucleotides in an RNAtranscript, such as in the 3′ untranslated region (3′-UTR), so as toregulate transcription, stability and/or translation of that RNAtranscript in a manner dependent on the ligand. To further illustrate,the expression construct can include a coding sequence for a polypeptidesuch that the mRNA transcript includes both the polypeptide codingsequence as well as one or more of the regulated polynucleotides of theinvention. In this way, expression of the polypeptide can be rendereddependent on the ligand to which the aptamer binds.

The invention further provides a class of in vivo nucleic acid sensors,for example, aptamer-regulated nucleic acids that directly sense thepresence or amount an intracellular molecule through changes in nucleicacid conformation upon ligand binding to the aptamer domain of anaptamer-regulated nucleic acid. For example, a ligand that interactswith the aptamer domain of an aptamer-regulated nucleic acid switches“on” the actuator domain of the aptamer-regulated nucleic acid. Theactivated actuator domain then targets a “reporter” molecule. Thereporter molecule is activated or repressed by its interaction with theactuator domain. The amount or activity of the reporter molecule,therefore, correlates with the amount or concentration of the ligand ofinterest. Exemplary reporter molecules include, without limitation,fluorescent reporter proteins such as green fluorescent protein (GFP orany of its art-recognized variants) or luciferase, enzymatic reporterssuch as alkaline phosphatase, or colorimetric reporters such as lacZ.

Antiswitches

An aptamer-regulated nucleic acid of the invention may comprise anactuator domain that comprises an antisense sequence and acts through anantisense mechanism for inhibiting expression of a target gene. As usedherein, such aptamer-regulated nucleic acids are also referred to as“antiswitches.” Antisense technologies have been widely utilized toregulate gene expression (Buskirk et al., Chem Biol 11, 1157-63 (2004);and Weiss et al., Cell Mol Life Sci 55, 334-58 (1999)). As used herein,“antisense” technology refers to administration or in situ generation ofmolecules or their derivatives which specifically hybridize (e.g., bind)under cellular conditions, with the target nucleic acid of interest(mRNA and/or genomic DNA) encoding one or more of the target proteins soas to inhibit expression of that protein, e.g., by inhibitingtranscription and/or translation, such as by steric hindrance, alteringsplicing, or inducing cleavage or other enzymatic inactivation of thetranscript. The binding may be by conventional base paircomplementarity, or, for example, in the case of binding to DNAduplexes, through specific interactions in the major groove of thedouble helix. In general, “antisense” technology refers to the range oftechniques generally employed in the art, and includes any therapy thatrelies on specific binding to nucleic acid sequences.

An aptamer-regulated nucleic acid that comprises an antisense effectordomain of the present invention can be delivered, for example, as acomponent of an expression plasmid which, when transcribed in the cell,produces an effector domain which is complementary to at least a uniqueportion of the target nucleic acid. Alternatively, the aptamer-regulatednucleic acid that comprises an antisense effector domain can begenerated outside of the target cell, and which, when introduced intothe target cell causes inhibition of expression by hybridizing with thetarget nucleic acid. Aptamer-regulated nucleic acids may be modified sothat they are resistant to endogenous nucleases, e.g. exonucleasesand/or endonucleases, and are therefore stable in vivo. Exemplarynucleic acid molecules for use in aptamer-regulated nucleic acids arephosphoramidate, phosphothioate and methylphosphonate analogs of DNA(see also U.S. Pat. Nos. 5,176,996; 5,264,564; and 5,256,775). Generalapproaches to constructing oligomers useful in antisense technology havebeen reviewed, for example, by van der Krol et al. (1988) Biotechniques6:958-976; and Stein et al. (1988) Cancer Res 48:2659-2668.

Several considerations may be taken into account when constructingantisense actuator domains for use in the compositions and methods ofthe invention: (1) antisense actuator domains preferably have a GCcontent of 50% or more; (2) generally, avoid sequences with stretches of3 or more Gs; and (3) antisense actuator domains preferably should notbe longer than 25-26 mers when in their “on” state and modulating atarget gene. When testing an antisense actuator domain, a mismatchedcontrol can be constructed. The controls can be generated by reversingor scrambling the sequence order of the corresponding antisenseoligonucleotide in order to conserve the same ratio of bases.

Antisense approaches involve the design of actuator domains (either DNAor RNA) that are complementary to a target nucleic acid encoding aprotein of interest. The antisense effector domain may bind to an mRNAtranscript and prevent translation of a protein of interest. Absolutecomplementarity, although preferred, is not required. In the case ofdouble-stranded antisense actuator domains, a single strand of theduplex DNA may thus be tested, or triplex formation may be assayed. Theability to hybridize will depend on both the degree of complementarityand the length of the antisense sequence. Generally, the longer thehybridizing nucleic acid, the more base mismatches with a target nucleicacid it may contain and still form a stable duplex (or triplex, as thecase may be). One skilled in the art can ascertain a tolerable degree ofmismatch by use of standard procedures to determine the melting point ofthe hybridized complex.

Antisense actuator domains that are complementary to the 5′ end of anmRNA target, e.g., the 5′ untranslated sequence up to and including theAUG initiation codon, should work most efficiently at inhibitingtranslation of the mRNA. However, sequences complementary to the 3′untranslated sequences of mRNAs have recently been shown to be effectiveat inhibiting translation of mRNAs as well (Wagner, R. 1994. Nature372:333). Therefore, antisense actuator domains complementary to eitherthe 5′ or 3′ untranslated, non-coding regions of a target gene could beused in an antisense approach to inhibit translation of a target mRNA.Antisense actuator domains complementary to the 5′ untranslated regionof an mRNA should include the complement of the AUG start codon.Antisense oligonucleotides complementary to mRNA coding regions are lessefficient inhibitors of translation but could also be used in accordancewith the invention. Whether designed to hybridize to the 5′, 3′, orcoding region of mRNA, antisense nucleic acids should be at least sixnucleotides in length, and are preferably less than about 100, and morepreferably less than about 50, 25, 17 or 10 nucleotides in length.

Regardless of the choice of target sequence, it is preferred that invitro studies are first performed to quantitate the ability of theantiswitch to inhibit expression of a target gene. It is preferred thatthese studies utilize controls that distinguish between antisense geneinhibition and nonspecific biological effects of antiswitches. It isalso preferred that these studies compare levels of the target RNA orprotein with that of an internal control RNA or protein. Additionally,it is envisioned that results obtained using the antiswitch are comparedwith those obtained using a control antiswitch. It is preferred that thecontrol antiswitch is of approximately the same length as the testantiswitch and that the nucleotide sequence of the control antiswitchdiffers from the antisense sequence of interest no more than isnecessary to prevent specific hybridization to the target sequence.

Antiswitches can be DNA or RNA or chimeric mixtures or derivatives ormodified versions thereof, single-stranded or double-stranded.Antiswitches can be modified at the base moiety, sugar moiety, orphosphate backbone, for example, to improve stability of the molecule,hybridization, etc. Antiswitches may include other appended groups suchas peptides (e.g., for targeting host cell receptors), or agentsfacilitating transport across the cell membrane (see, e.g., Letsinger etal., 1989, Proc Natl Acad. Sci. USA 86:6553-6556; Lemaitre et al., 1987,Proc Natl Acad. Sci. USA 84:648-652; PCT Publication No. WO88/09810,published Dec. 15, 1988) or the blood-brain barrier (see, e.g., PCTPublication No. WO89/10134, published Apr. 25, 1988),hybridization-triggered cleavage agents. (See, e.g., Krol et al., 1988,BioTechniques 6:958-976) or intercalating agents. (See, e.g., Zon,Pharm. Res. 5:539-549 (1988)). To this end, an antiswitch may beconjugated to another molecule, e.g., a peptide, hybridization triggeredcross-linking agent, transport agent, hybridization-triggered cleavageagent, etc.

An antiswitch may comprise at least one modified base moiety which isselected from the group including but not limited to 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxytriethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil; beta-D-mannosylqueosine,5-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

An antiswitch may also comprise at least one modified sugar moietyselected from the group including but not limited to arabinose,2-fluoroarabinose, xylulose, and hexose.

An antiswitch can also contain a neutral peptide-like backbone. Suchmolecules are termed peptide nucleic acid (PNA)-oligomers and aredescribed, e.g., in Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci.USA 93:14670 and in Eglom et al. (1993) Nature 365:566. One advantage ofPNA oligomers is their capability to bind to complementary DNAessentially independently from the ionic strength of the medium due tothe neutral backbone of the DNA. In yet another embodiment, anantiswitch comprises at least one modified phosphate backbone selectedfrom the group consisting of a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

In a further embodiment, an antiswitch is an anomeric oligonucleotide.An anomeric oligonucleotide forms specific double-stranded hybrids withcomplementary RNA in which, contrary to the usual units, the strands runparallel to each other (Gautier et al., 1987, Nucl. Acids Res.15:6625-6641). The oligonucleotide is a 2′-O-methylribonucleotide (Inoueet al., 1987, Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNAanalogue (Inoue et al., 1987, FEBS Lett. 215:327-330).

Aptamer-regulated nucleic acids of the invention, includingantiswitches, may be synthesized by standard methods known in the art,e.g., by use of an automated DNA synthesizer (such as are commerciallyavailable from Biosearch, Applied Biosystems, etc.). As examples,phosphorothioate oligonucleotides may be synthesized by the method ofStein et al. Nucl. Acids Res. 16:3209 (1988)), methylphosphonateoligonucleotides can be prepared by use of controlled pore glass polymersupports (Sarin et al., Proc. Natl. Acad. Sci. USA 85:7448-7451 (1988)),etc.

While antisense sequences complementary to the coding region of an mRNAsequence can be used, those complementary to the transcribeduntranslated region and to the region comprising the initiatingmethionine are most preferred.

Antiswitch nucleic acid molecules can be delivered to cells that expresstarget genes in vivo. A number of methods have been developed fordelivering nucleic acids into cells; e.g., they can be injected directlyinto the tissue site, or modified nucleic acids, designed to target thedesired cells (e.g., antiswitches linked to peptides or antibodies thatspecifically bind receptors or antigens expressed on the target cellsurface) can be administered systematically.

However, it may be difficult to achieve intracellular concentrations ofthe antiswitch sufficient to attenuate the activity of a target gene ormRNA or interest in certain instances. Therefore, another approachutilizes a recombinant DNA construct in which the antiswitch or otheraptamer-regulated nucleic acid is placed under the control of a strongpol III or pol II promoter. The use of such a construct to transfecttarget cells in the patient will result in the transcription ofsufficient amounts of antiswitches that will form complementary basepairs with the target gene or mRNA and thereby attenuate the activity ofthe protein of interest. For example, a vector can be introduced in vivosuch that it is taken up by a cell and directs the transcription of anantiswitch. Such a vector can remain episomal or become chromosomallyintegrated, as long as it can be transcribed to produce the desiredantiswitch. Such vectors can be constructed by recombinant DNAtechnology methods standard in the art. Vectors can be plasmid, viral,or others known in the art, used for replication and expression inmammalian cells. A promoter may be operably linked to the sequenceencoding the antiswitch. Expression of the sequence encoding theantiswitch can be by any promoter known in the art to act in mammalian,preferably human cells. Such promoters can be inducible or constitutive.Such promoters include but are not limited to: the SV40 early promoterregion (Bernoist and Chambon, Nature 290:304-310 (1981)), the promotercontained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamotoet al., Cell 22:787-797 (1980)), the herpes thymidine kinase promoter(Wagner et al., Proc. Natl. Acad. Sci. USA 78:1441-1445 (1981)), theregulatory sequences of the metallothionine gene (Brinster et al.,Nature 296:3942 (1982)), etc. Any type of plasmid, cosmid, YAC or viralvector can be used to prepare the recombinant DNA construct that can beintroduced directly into the tissue site. Alternatively, viral vectorscan be used which selectively infect the desired tissue, in which caseadministration may be accomplished by another route (e.g.,systematically).

RNAi-Based Switch Constructs (with siRNA or miRNA Actuator Domains)

RNA interference (RNAi) is a phenomenon describing double-stranded(ds)RNA-dependent gene specific posttranscriptional silencing. Initialattempts to harness this phenomenon for experimental manipulation ofmammalian cells were foiled by a robust and nonspecific antiviraldefense mechanism activated in response to long dsRNA molecules. Gil etal. Apoptosis 2000, 5:107-114. The field was significantly advanced uponthe demonstration that synthetic duplexes of 21 nucleotide RNAs couldmediate gene specific RNAi in mammalian cells, without invoking genericantiviral defense mechanisms. Elbashir et al. Nature 2001, 411:494-498;Caplen et al. Proc Natl Acad Sci 2001, 98:9742-9747. As a result, siRNAsand miRNAs have become powerful tools to dissect gene function. Thechemical synthesis of small RNAs is one avenue that has producedpromising results. Numerous groups have also sought the development ofDNA-based vectors capable of generating such siRNA within cells. Severalgroups have recently attained this goal and published similar strategiesthat, in general, involve transcription of short hairpin (sh)RNAs thatare efficiently processed to form siRNAs within cells. Paddison et al.PNAS 2002, 99:1443-1448; Paddison et al. Genes & Dev 2002, 16:948-958;Sui et al. PNAS 2002, 8:5515-5520; and Brummelkamp et al. Science 2002,296:550-553. These reports describe methods to generate siRNAs capableof specifically targeting numerous endogenously and exogenouslyexpressed genes.

Accordingly, the present invention provides an aptamer-regulated nucleicacid comprising an actuator domain that comprises an RNAi sequence andacts through an RNAi or miRNA mechanism to attenuate expression of atarget gene. For instance, an aptamer-regulated nucleic acid maycomprise an actuator domain that comprises a miRNA or siRNA sequence orprecursor thereof. In one embodiment, an actuator domain comprises amiRNA or siRNA sequence that is between about 19 nucleotides and about75 nucleotides in length, or preferably, between about 25 base pairs andabout 35 base pairs in length. In certain embodiments, the actuatordomain is a hairpin loop that may be processed by RNase III enzymes(e.g., Drosha and Dicer).

An RNAi construct contains a nucleotide sequence that hybridizes underthe physiologic conditions of the cell to the nucleotide sequence of atleast a portion of the mRNA transcript for the gene to be inhibited(i.e., the “target” gene). The double-stranded RNA only needs to besufficiently similar to natural RNA for its ability to mediate RNAi.Thus, the invention has the advantage of being able to tolerate sequencevariations that might be expected due to genetic mutation, strainpolymorphism or evolutionary divergence. The number of toleratednucleotide mismatches between the target sequence and the RNAi constructsequence is no more than 1 in 5 basepairs, or 1 in 10 basepairs, or 1 in20 basepairs, or 1 in 50 basepairs. Mismatches in the center of thesiRNA duplex are most critical and may abolish cleavage of the targetRNA. In contrast, nucleotides at the 3′ end of the siRNA strand that iscomplementary to the target RNA do not significantly contribute tospecificity of the target recognition. However, certain miRNA designs,such as the mir-30 based miRNA designs, may feature a bulge of about afew nucleotides in the middle of the guide sequence.

Sequence identity may be optimized by sequence comparison and alignmentalgorithms known in the art (see Gribskov and Devereux, SequenceAnalysis Primer, Stockton Press, 1991, and references cited therein) andcalculating the percent difference between the nucleotide sequences by,for example, the Smith-Waterman algorithm as implemented in the BESTFITsoftware program using default parameters (e.g., University of WisconsinGenetic Computing Group). Greater than 90% sequence identity, or even100% sequence identity, between the inhibitory RNA and the portion ofthe target gene is preferred. Alternatively, the duplex region of theRNA may be defined functionally as a nucleotide sequence that is capableof hybridizing with a portion of the target gene transcript (e.g., 400mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. hybridizationfor 12-16 hours; followed by washing).

Production of aptamer-regulated nucleic acids that comprise an actuatordomain comprising RNAi sequences can be carried out by any of themethods for producing aptamer-regulated nucleic acids described herein.For example, an aptamer-regulated nucleic acid can be produced bychemical synthetic methods or by recombinant nucleic acid techniques.Endogenous RNA polymerase of the treated cell may mediate transcriptionin vivo, or cloned RNA polymerase can be used for transcription invitro. Aptamer-regulated nucleic acids, including antiswitches or thosethat modulate target gene activity by RNAi mechanisms may includemodifications to either the phosphate-sugar backbone or the nucleoside,e.g., to reduce susceptibility to cellular nucleases, improvebioavailability, improve formulation characteristics, and/or changeother pharmacokinetic properties. For example, the phosphodiesterlinkages of natural RNA may be modified to include at least one of anitrogen or sulfur heteroatom. Modifications in RNA structure may betailored to allow specific genetic inhibition while avoiding a generalresponse to dsRNA. Likewise, bases may be modified to block the activityof adenosine deaminase. Aptamer-regulated nucleic acids may be producedenzymatically or by partial/total organic synthesis, any modifiedribonucleotide can be introduced by in vitro enzymatic or organicsynthesis.

Methods of chemically modifying RNA molecules can be adapted formodifying RNAi constructs (see, for example, Heidenreich et al. (1997)Nucleic Acids Res, 25:776-780; Wilson et al. (1994) J Mol Recog 7:89-98;Chen et al. (1995) Nucleic Acids Res 23:2661-2668; Hirschbein et al.(1997) Antisense Nucleic Acid Drug Dev 7:55-61). Merely to illustrate,the backbone of an RNAi construct can be modified withphosphorothioates, phosphoramidate, chimericmethylphosphonate-phosphodiesters, phosphodithioates, peptide nucleicacids, 5-propynyl-pyrimidine containing oligomers or sugar modifications(e.g., 2′-substituted ribonucleosides, a-configuration).

The double-stranded structure may be formed by a singleself-complementary RNA strand or two complementary RNA strands. RNAduplex formation may be initiated either inside or outside the cell. TheRNA may be introduced in an amount which allows delivery of at least onecopy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000copies per cell) of double-stranded material may yield more effectiveinhibition, while lower doses may also be useful for specificapplications. Inhibition is sequence-specific in that nucleotidesequences corresponding to the duplex region of the RNA are targeted forgenetic inhibition.

In certain embodiments, the subject RNAi constructs are “siRNAs.” Thesenucleic acids are between about 19-35 nucleotides in length, and evenmore preferably 21-23 nucleotides in length, e.g., corresponding inlength to the fragments generated by nuclease “dicing” of longerdouble-stranded RNAs. The siRNAs are understood to recruit nucleasecomplexes and guide the complexes to the target mRNA by pairing to thespecific sequences. As a result, the target mRNA is degraded by thenucleases in the protein complex or translation is inhibited. In aparticular embodiment, the 21-23 nucleotides siRNA molecules comprise a3′ hydroxyl group.

In other embodiments, the subject RNAi constructs are “miRNAs.”microRNAs (miRNAs) are small non-coding RNAs that direct posttranscriptional regulation of gene expression through interaction withhomologous mRNAs. miRNAs control the expression of genes by binding tocomplementary sites in target mRNAs from protein coding genes. miRNAsare similar to siRNAs. miRNAs are processed by nucleolytic cleavage fromlarger double-stranded precursor molecules. These precursor moleculesare often hairpin structures of about 70 nucleotides in length, with 25or more nucleotides that are base-paired in the hairpin. The RNaseIII-like enzymes Drosha and Dicer (which may also be used in siRNAprocessing) cleave the miRNA precursor to produce an miRNA. Theprocessed miRNA is single-stranded and incorporates into a proteincomplex, termed RISC or miRNP. This RNA-protein complex targets acomplementary mRNA. miRNAs inhibit translation or direct cleavage oftarget mRNAs. (Brennecke et al., Genome Biology 4:228 (2003); Kim etal., Mol. Cells. 19:1-15 (2005).

In certain embodiments, miRNA and siRNA constructs can be generated byprocessing of longer double-stranded RNAs, for example, in the presenceof the enzymes Dicer or Drosha. Dicer and Drosha are RNAse III-likenucleases that specifically cleave dsRNA. Dicer has a distinctivestructure which includes a helicase domain and dual RNAse III motifs.Dicer also contains a region of homology to the RDE1/QDE2/Argonautefamily, which have been genetically linked to RNAi in lower eukaryotes.Indeed, activation of, or overexpression of Dicer may be sufficient inmany cases to permit RNA interference in otherwise non-receptive cells,such as cultured eukaryotic cells, or mammalian (non-oocytic) cells inculture or in whole organisms. Methods and compositions employing Dicer,as well as other RNAi enzymes, are described in U.S. Pat. App.Publication No. 20040086884.

In one embodiment, the Drosophila in vitro system is used. In thisembodiment, an aptamer-regulated nucleic acid is combined with a solubleextract derived from Drosophila embryo, thereby producing a mixture,which is maintained under conditions in which the dsRNA is processed toRNA molecules of about 21 to about 23 nucleotides.

The miRNA and siRNA molecules can be purified using a number oftechniques known to those of skill in the art. For example, gelelectrophoresis can be used to purify such molecules. Alternatively,non-denaturing methods, such as non-denaturing column chromatography,can be used to purify the siRNA and miRNA molecules. In addition,chromatography (e.g., size exclusion chromatography), glycerol gradientcentrifugation, affinity purification with antibody can be used topurify siRNAs and miRNAs.

In certain preferred embodiments, at least one strand of the siRNAsequence of an effector domain has a 3′ overhang from about 1 to about 6nucleotides in length, though may be from 2 to 4 nucleotides in length.More preferably, the 3′ overhangs are 1-3 nucleotides in length. Incertain embodiments, one strand having a 3′ overhang and the otherstrand being blunt-ended or also having an overhang. The length of theoverhangs may be the same or different for each strand. In order tofurther enhance the stability of the siRNA sequence, the 3′ overhangscan be stabilized against degradation. In one embodiment, the RNA isstabilized by including purine nucleotides, such as adenosine orguanosine nucleotides. Alternatively, substitution of pyrimidinenucleotides by modified analogues, e.g., substitution of uridinenucleotide 3′ overhangs by 2′-deoxythyinidine is tolerated and does notaffect the efficiency of RNAi. The absence of a 2′ hydroxylsignificantly enhances the nuclease resistance of the overhang in tissueculture medium and may be beneficial in vivo.

In certain embodiments, an aptamer-regulated nucleic acid is in the formof a hairpin structure (named as hairpin RNA). The hairpin RNAs can besynthesized exogenously or can be formed by transcribing from RNApolymerase II or III promoters in vivo. Examples of making and usingsuch hairpin RNAs for gene silencing in mammalian cells are describedin, for example, Paddison et al., Genes Dev, 2002, 16:948-58; McCaffreyet al., Nature, 2002, 418:38-9; McManus et al., RNA 2002, 8:842-50; Yuet al., Proc Natl Acad Sci USA, 2002, 99:6047-52). Preferably, suchhairpin RNAs are engineered in cells or in an animal to ensurecontinuous and stable suppression of a desired gene. It is known in theart that miRNAs and siRNAs can be produced by processing a hairpin RNAin the cell.

In yet other embodiments, a plasmid is used to deliver thedouble-stranded RNA, e.g., as a transcriptional product. After thecoding sequence is transcribed, the complementary RNA transcriptsbase-pair to form the double-stranded RNA.

4. Exemplary Formulations

The aptamer-regulated nucleic acids of the invention may also beadmixed, encapsulated, conjugated or otherwise associated with othermolecules, molecule structures or mixtures of compounds, as for example,liposomes, polymers, receptor targeted molecules, oral, rectal, topicalor other formulations, for assisting in uptake, distribution and/orabsorption. The subject aptamer-regulated nucleic acids can be providedin formulations also including penetration enhancers, carrier compoundsand/or transfection agents.

Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption assisting formulations which canbe adapted for delivery of switch molecules include, but are not limitedto, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127;5,521,291; 51,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330;4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221;5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854;5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and5,595,756.

Aptamer-regulated nucleic acids of the invention also encompass anypharmaceutically acceptable salts, esters or salts of such esters, orany other compound which, upon administration to an animal including ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to aptamer-regulated nucleic acids andpharmaceutically acceptable salts, and other bioequivalents.

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Examples of metals used as cations are sodium potassium, magnesium,calcium, and the like. Examples of suitable amines areN,NI-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci., 1977, 66, 1-19). The base addition salts of the acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a “pharmaceutical addition salt”includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Preferred acid salts arethe hydrochlorides, acetates, salicylates, nitrates and phosphates.Other suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids. Preferred examples of pharmaceutically acceptable saltsinclude but are not limited to (a) salts formed with cations such assodium, potassium, ammonium, magnesium, calcium, polyamines such asspermine and spermidine, etc.; (b) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; (c) saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) saltsformed from elemental anions such as chlorine, bromine, and iodine.

Other formulations, delivery methods, and routes of administration arealso provided, such as those described in U.S. Pat. App. Publication No.20040063654.

5. Exemplary Uses

One aspect of the invention provides a method of modulating the amountand/or activity of a ligand in a cell. The method may comprise designingand selecting an aptamer responsive to the ligand and providing anaptamer-regulated nucleic acid comprising the selected aptamer and aneffector RNA, and the effector RNA is targeted to a molecule, asignaling and/or metabolic pathway in the cell that involves the ligand,for example, as a metabolite or an intermediate molecule (or theligand-associated signaling and/or metabolic pathway). The method mayfurther comprise contacting a cell with the aptamer-regulated nucleicacid in an amount and/or for a time period sufficient for modulating theconcentration and/or activity of the ligand in the cell when switched“on.”

Also provided is a method of modulating a biological or biochemicalresponse of a cell to the presence, amount and/or activity of a ligandin a cell. The method may comprise designing and selecting an aptamerresponsive to the ligand and providing an aptamer-regulated nucleic acidcomprising the selected aptamer and an effector RNA, and the effectorRNA is targeted to a gene that modulates a biological or biochemicalresponse of the cell to the presence, amount and/or activity of theligand in the cell. The method may further comprise contacting a cellwith the aptamer-regulated nucleic acid in an amount and/or for a timeperiod sufficient for modulating the biological or biochemical responseof the cell when switched “on.”

Certain embodiments are also directed to a method of establishing aconditional genetic network, as described herein. The method maycomprise providing an aptamer-regulated nucleic acid that comprises anaptamer domain and an effector domain; the aptamer domain is responsiveto a ligand, while the effector domain is targeted to a molecule that isindependent of the ligand-associated signaling pathway. The methodfurther comprises contacting the cell with the aptamer-regulated nucleicacid in an effective amount and/or for a sufficient time period, whenswitched “on,” that modulates expression of the target molecule, therebyestablishing a conditional genetic network.

In a further aspect, a method of the invention is used to inhibit, or atleast reduce, unwanted growth of cells in vivo, and particularly thegrowth of transformed cells. In certain embodiments, the subject methodutilizes one or more aptamer-regulated nucleic acids of the invention toselectively inhibit the expression of genes encodingproliferation-regulating proteins. For instance, the subject method canbe used to inhibit expression of a gene product that is essential tomitosis in the target cell, and/or which is essential to preventingapoptosis of the target cell. The aptamer-regulated nucleic acids of thepresent invention, in particular, the effector domains, can be designedto correspond to the coding sequence or other portions of mRNAs encodingthe targeted proliferation-regulating protein. When treated with theaptamer-regulated nucleic acid, the loss-of-expression phenotype whichresults in the target cell causes the cell to become quiescent or toundergo apoptosis.

In certain embodiments, the subject aptamer-regulated nucleic acids areselected to inhibit expression of gene products which stimulate cellgrowth and mitosis. One class of genes which can be targeted by themethod of the present invention are those known as oncogenes. As usedherein, the term “oncogene” refers to a gene which stimulates cellgrowth and, when its level of expression in the cell is reduced, therate of cell growth is reduced or the cell becomes quiescent. In thecontext of the present invention, oncogenes include intracellularproteins, as well as extracellular growth factors which may stimulatecell proliferation through autocrine or paracrine function. Examples ofhuman oncogenes against which switch molecules can designed includec-myc, c-myb, mdm2, PKA-I (protein kinase A type I), Abl-1, Bcl2, Ras,c-Raf kinase, CDC25 phosphatases, cyclins, cyclin dependent kinases(cdks), telomerase, PDGF/sis, erb-B, fos, jun, mos, and src, to name buta few. In the context of the present invention, oncogenes also include afusion gene resulted from chromosomal translocation, for example, theBcr/Abl fusion oncogene.

In certain embodiments, the cell is a transformed cell so that theaptamer-regulated nucleic acid is used for the treatment of hyperplasticcell growth, including treatment of a cancer. In other embodiments, theaptamer-regulated nucleic acid is used for inhibiting activation oflymphocytes, including treatment or prophylaxis of immune-mediatedinflammatory disorders. In still other embodiments, theaptamer-regulated nucleic acid is used for inhibiting proliferation ofsmooth muscle cells, including treatment or prophylaxis of restenosis.In yet other embodiments, the aptamer-regulated nucleic acid is used forinhibiting proliferation of epithelial cells (e.g., as a component ofcosmetic preparations).

In certain preferred embodiments, the subject aptamer-regulated nucleicacids are selected by their ability to inhibit expression of a gene(s)essential for proliferation of a transformed cell, and particularly of atumor cell. Such that the aptamer-regulated nucleic acids can be used aspart of the treatment or prophylaxis for neoplastic, anaplastic and/orhyperplastic cell growth in vivo, including as part of a treatment of atumor. The c-myc protein is deregulated in many forms of cancer,resulting in increased expression. Reduction of c-myc RNA levels invitro results in induction of apoptosis. An antisense, siRNA, miRNA, orRNAi effector domain complementary to c-myc can therefore potentially beused as therapeutic for anti-cancer treatment. Preferably, the subjectaptamer-regulated nucleic acids can be used in the therapeutic treatmentof chronic lymphatic leukemia. Chronic lymphatic leukemia is oftencaused by a translocation of chromosomes 9 and 12 resulting in a Bcr/Ablfusion product. The resulting fusion protein acts as an oncogene;therefore, specific elimination of Bcr/Abl fusion mRNA may result incell death in the leukemia cells. Indeed, transfection of siRNAmolecules specific for the Bcr/Abl fusion mRNA into cultured leukemiccells, not only reduced the fusion mRNA and corresponding oncoprotein,but also induced apoptosis of these cells (see, for example, Wilda etal., Oncogene, 2002, 21:5716-5724).

In other embodiments, the subject aptamer-regulated nucleic acids areselected by their ability to inhibit expression of a gene(s) essentialfor activation of lymphocytes, e.g., proliferation of B-cells orT-cells, and particularly of antigen-mediated activation of lymphocytes.Such aptamer-regulated nucleic acids can be used as immunosuppressantagents, e.g., as part of the treatment or prophylaxis forimmune-mediated inflammatory disorders.

Alternatively, in other embodiments, the subject aptamer-regulatednucleic acids are selected by their ability to regulate activation ofimmune cells in response to particular exogenous or endogenous signals(such as cytokines or small molecules) in, for example, cell-basedimmunotherapy applications.

In certain embodiments, the methods described herein can be employed forthe treatment of autoimmune disorders. For example, the subjectaptamer-regulated nucleic acids are selected for their ability toinhibit expression of a gene(s) which encode or regulate the expressionof cytokines. Accordingly, constructs that cause inhibited or decreasedexpression of cytokines such as TNF-alpha, IL-1 alpha, IL-6 or IL-12, ora combination thereof, can be used as part of a treatment or prophylaxisfor rheumatoid arthritis. Similarly, constructs that cause inhibited ordecreased expression of cytokines involved in inflammation can be usedin the treatment or prophylaxis of inflammation and inflammation-relateddiseases, such as multiple sclerosis.

In other embodiments, the subject aptamer-regulated nucleic acids areselected for their ability to inhibit expression of a gene(s) implicatedin the onset or progression of diabetes. For example, experimentaldiabetes mellitus was found to be related to an increase in expressionof p21WAF1/CIP1 (p21), and TGF-beta 1 has been implicated in glomerularhypertrophy (see, for example, Al-Douahji, et al. Kidney Int.56:1691-1699). Accordingly, constructs that cause inhibited or decreasedexpression of these proteins can be used in the treatment or prophylaxisof diabetes.

In other embodiments, the subject aptamer-regulated nucleic acids areselected for their ability to inhibit expression of ICAM-1(intracellular adhesion molecule). An antisense nucleic acid thatinhibits expression of ICAM-1 is being developed by Isis pharmaceuticsfor psoriasis. Additionally, an antisense nucleic acid against theICAM-1 gene is suggested for preventing acute renal failure andreperfusion injury and for prolonging renal isograft survival (see, forexample, Halier et al. (1996) Kidney Int. 50:473-80; Dragun et al.(1998) Kidney Int. 54:590-602; Dragun et al. (1998) Kidney Int.54:2113-22). Accordingly, the present invention contemplates the use ofaptamer-regulated nucleic acids comprising similar antisense effectorRNA domains, siRNA, miRNA, or RNAi effector domains targeting ICAM-1gene in the above-described diseases.

In other embodiments, the subject aptamer-regulated nucleic acids areselected by their ability to inhibit expression of a gene(s) essentialfor proliferation of smooth muscle cells or other cells of endotheliumof blood vessels, such as proliferating cells involved in neointimaformation. In such embodiments, the subject method can be used as partof a treatment or prophylaxis for restenosis.

Merely to illustrate, aptamer-regulated nucleic acids applied to theblood vessel endothelial cells after angioplasty can reduceproliferation of these cells after the procedure. Merely to illustrate,a specific example is an siRNA complementary to c-myc (an oncogene).Down-regulation of c-myc inhibits cell growth. Therefore, an effectordomain comprising an siRNA sequence can be prepared by including thefollowing sequence in an effector domain:

5′-UCCCGCGACGAUGCCCCUCATT-3′ 3′-TTAGGGCGCUGCUACGGGGAGU-5′

All bases are ribonucleic acids except the thymidines shown in bold,which are deoxyribose nucleic acids (for more stability).Double-stranded RNA can be prepared by mixing the oligonucleotides atequimolar concentrations in 10 mM Tris-Cl (pH 7.0) and 20 mM NaCl,heating to 95° C., and then slowly cooling to 37° C. Alternatively, thesequence can be included in a hairpin structure. The resulting nucleicacid can then be purified by agarose gel electrophoresis and deliveredto cells either free or complexed to a delivery system such as acyclodextrin-based polymer. For in vitro experiments, the effect of theaptamer-regulated nucleic acid can be monitored by growth curveanalysis, RT-PCR or western blot analysis for the c-myc protein.

It is demonstrated that antisense oligodeoxynucleotides directed againstthe c-myc gene inhibit restenosis when given by local deliveryimmediately after coronary stent implantation (see, for example, Kutryket al. (2002) J Am Coll Cardiol. 39:281-287; Kipshidze et al. (2002) JAm Coll Cardiol. 39:1686-1691). Therefore, the present inventioncontemplates delivering an aptamer-regulated nucleic acid against thec-Myc gene (i.e., c-Myc antisense or RNAi construct) to the stentimplantation site with an infiltrator delivery system (InterventionalTechnologies, San Diego, Calif.). Preferably, the c-Myc-targetingaptamer-regulated nucleic acid is directly coated on stents forinhibiting restenosis. Similarly, the c-Myc-targeting aptamer-regulatednucleic acid can be delivered locally for inhibiting myointimalhyperplasia after percutaneous transluminal coronary angioplasty (PTCA)and exemplary methods of such local delivery can be found, for example,Kipshidze et al. (2001) Catheter Cardiovasc Interv. 54:247-56. Incertain embodiments, the aptamer-regulated nucleic acids are chemicallymodified with, for example, phosphorothioates or phosphoramidate.

Early growth response factor-1 (i.e., Egr-1) is a transcription factorthat is activated during mechanical injury and regulates transcriptionof many genes involved with cell proliferation and migration. Therefore,down-regulation of this protein may also be an approach for preventionof restenosis. An effector domain of an aptamer-regulated nucleic aciddirected against the Egr-1 gene can be prepared by including thefollowing sequence in the effector domain:

5′-UCGUCCAGGAUGGCCGCGGTT-3′ 3′-TTAGCAGGUCCUACCGGCGCC-5′

Again, all bases are ribonucleic acids except the thymidines shown inbold, which are deoxyribose nucleic acids. The effector domains andthereby the aptamer-regulated nucleic acids can be prepared from thesesequences and introduced into cells as described herein.

A further aspect of invention relates to applications of a ligandcontrolled nucleic acid molecule in different fields. For example, anaptamer-regulated nucleic acid can be employed to detect the presence orabsence or the amount of a target molecule in a sample. The targetmolecule may be a metabolite, an ion, a peptide, a nucleic acid, etc. Anaptamer-regulated nucleic acid can similarly be employed for imagingpurposes. Further, an aptamer-regulated nucleic acid can be employed totarget the effector nucleic acid domain to certain environments, e.g., aparticular intracellular location or cell membrane.

An aptamer-regulated nucleic acid of the invention can function as atool to sense and detect metabolite levels in a cell, which can benoninvasive. The imaging or detection can be used for quantification ofone or more metabolites of interest. Alternatively, it can be used toregulate certain enzymes in a signaling pathway for control over fluxthrough a pathway and product formation or to alter that metabolic stateby targeting levels of proteins or enzymes in a cell.

In one embodiment, the invention provides a method of modulating theconcentration and/or activity of a ligand in a cell. The method maycomprise designing and selecting an aptamer responsive to the ligand andproviding an aptamer-regulated nucleic acid comprising the selectedaptamer and an effector RNA, wherein the effector RNA is targeted to amolecule, a signaling pathway, or a metabolic pathway in a cell thatinvolves the ligand (e.g., a metabolite or an intermediate molecule).The methods of the invention may be used, for example, to respond to theaccumulation of a toxic intermediate, to attenuate the activity of asignaling or metabolic pathway, or to alter the growth, survival, ordifferentiation of treated cells.

In another embodiment, a method is provided for modulating theconcentration and/or activity of a target gene that is in a differentsignaling and/or metabolic pathway than that of the ligand. According tothis method, aptamer-regulated nucleic acids may be used to establishconditional genetic networks. The method may comprise designing andselecting an aptamer responsive to a ligand and providing anaptamer-regulated nucleic acid comprising the selected aptamer and aneffector RNA that is targeted to a molecule, a signaling pathway, or ametabolic pathway in a cell that is independent of the ligand-associatedsignaling pathway. Accordingly, aptamer-regulated nucleic acids may beused to engineer intracellular gene networks by sensing endogenouslygenerated signals (e.g., ligands) and responding to these signals byaffecting the expression of a gene in signaling pathways independent ofthe ligand. Aptamer-regulated nucleic acids may be used to allow cellsto appropriately respond to the buildup of toxic intermediates andcompounds, or to alter the physiology of a cell (e.g., growth, survival,or differentiation).

In a related embodiment, the methods and compositions of the inventionmay be adapted to monitor the concentration of metabolites. The methodmay comprise a sensor aptamer-regulated nucleic acid wherein the aptamerdomain of the switch is responsive to the metabolite. By measuringchanges in metabolite concentrations, aptamer-regulated nucleic acidsmay be used to determine the full range of biochemical effects induced,for example, by a therapeutic intervention. Aptamer-regulated nucleicacids can further be used to diagnose or predict disease by monitoringmetabolite concentrations.

In other embodiments, aptamer-regulated nucleic acids can serve as toolsfor interfacing with the environment including both intracellularenvironment and extracellular environment. For example, anaptamer-regulated nucleic acid can be used to “transport” a target RNAto the cell membrane or other cellular locations, potentially by theaptamer domain that recognizes a signal peptide, and alternatively, aparticular target.

EXAMPLES

Having generally described the invention, Applicants refer to thefollowing illustrative examples to help to understand the generallydescribed invention. These specific examples are included merely toillustrate certain aspects and embodiments of the present invention, andthey are not intended to limit the invention in any respect. Certaingeneral principles described in the examples, however, may be generallyapplicable to other aspects or embodiments of the invention.

Progress in constructing biological networks will rely on thedevelopment of more advanced components that can be predictably modifiedto yield optimal system performance. Applicants have engineered anRNA-based platform (“shRNA switch” as used herein) that provides forintegrated ligand control of RNA interference (RNAi) by modular couplingof an aptamer, switching strand, and small hairpin (sh)RNA stem into asingle component that links ligand concentration and target geneexpression levels. The follow Examples 1-9 illustrate a combinedexperimental and mathematical modeling approach that have identifiedmultiple tuning strategies, and moves toward a predictable framework forthe forward rational design of switches, such as shRNA switches. Theutility of this general platform is highlighted by the demonstration offine tuning, and model-guided design of shRNA switches with an optimizeddynamic range, although other switches based on ribozyme or antisensesequences can also be designed based on the same principle with minormodification. Thus, such switches can serve as an advanced component forthe construction of complex biological systems and offer a controlledmeans of activating various actuator domains (such as RNAi substrates)in disease therapeutics.

Example 1 Design and Characterization of a Modular shRNA Switch Platform

Applicants have developed a framework for the construction of shRNAswitches that mediate ligand control of RNAi across diverse mammaliancell types. The platform utilizes a strand displacement strategy, wherethe functions of ligand binding, translation of the binding interactioninto reduced processing by the RNAi machinery, and RNAi activation areisolated to individual domains, which demonstrates the generality of themethod in other switch platforms (e.g., ribozyme-based switch andantisense-based switch, etc.), and the ease of successful domainswapping and subsequent broad application in these switch platforms.

In addition, Applicants used the shRNA switch platform as a model tosystematically investigate tunability of the switch transfer functionthrough a combined experimental and mathematical modeling approach thatresulted in the identification of five generally applicable tuningstrategies. Standard RNA folding algorithms (Mathews et al, 2004) wereemployed to establish a quantitative sequence-to-function relationship.These efforts highlight the current limitations of these broadly-usedalgorithms for the design of RNAs that function in vivo and offer aframework for optimizing RNA switch behavior in silico. By demonstratingcombinatorial tuning strategies, and model-guided forward design ofshRNA switches with an optimized dynamic range within a specifiedcontext, Applicants show that polynucleotide switches (such as the shRNAswitch used in these experiments) can be used in the construction ofcomplex biological systems by modularly and rationally combining thenumber of available components. The resulting switches can be used as aregulatory tool for gene expression.

Applicants engineered a complex RNAi substrate that encodes aligand-controlled gene regulatory function by replacing the loop of asmall hairpin (sh)RNA with two domains: an aptamer and a switchingstrand (FIG. 1A). A competing strand (not marked), continuous with theswitching strand, forms part of the shRNA stem. The shRNA switchmolecule is designed to adopt distinct “active” and “inactive”conformations due to complementarity between the switching strand andthe shRNA stem. In the active conformation, irreversible processing bythe RNAi machinery of the formed shRNA stem results in small interfering(si)RNA production and subsequent RNAi-mediated silencing of the targetgene. In the inactive conformation, base-pairing by the switching stranddisrupts the shRNA stem, which is predicted to inhibit processing by theRNAi machinery (Macrae et al, 2006; Zeng and Cullen, 2004). Thisbase-pairing coincides with formation of the aptamer domain, such thatligand binding stabilizes the inactive conformation and indirectlyreduces siRNA production, thereby linking intracellular ligandconcentration to target protein levels through a component transferfunction. To decrease the activation energy separating the twoconformations, Applicants removed two nucleotides in the passengerstrand, thereby mimicking the bulge from the microRNA (miRNA) mir-30a(Griffiths-Jones, 2004; Griffiths-Jones et al, 2006).

The three domains that comprise an shRNA switch perform distinctfunctions: the shRNA stem encodes the guide strand that activatesRNAi-mediated silencing of the target gene, the aptamer detects themolecular input concentration through a ligand-binding interaction, andthe switching strand translates the binding interaction into a decreasein regulatory activity by affecting processing by the RNAi machinery.Based on the action of the switching strand that is complementary to theshRNA stem, the sequences of the shRNA stem and aptamer domains areindependent of one another. Therefore, the shRNA stem and aptamerdomains can be independently modified without altering the functionalityof the opposing domain or requiring sequence reassignment.

Applicants designed an initial shRNA switch (S1) to target EGFP andrespond to theophylline by incorporating an EGFP-targeting guide strandand the theophylline aptamer (Zimmermann et al, 2000) into our switchplatform (FIG. 1A). Applicants used in-line probing (Soukup and Breaker,1999, incorporated herein by reference, and briefly described below) toassess the structural characteristics of a T7-transcribed variantsimilar to S1 (S4t; FIG. 1B). In-line probing provides structuralinformation from the ligand dependence of spontaneous RNA cleavage. SeeMaterials and Methods section below.

For increasing theophylline concentrations, stabilization of the aptamerdomain coincided with destabilization of the switching strand and thedownstream shRNA stem sequence. The results suggest that theophyllinebinding promotes structural changes within and outside of the aptamerdomain. The apparent dissociation constant (K_(D)) of ˜5 μM, which wasdetermined by quantifying the cleavage products at two positions (FIG.7), is an order-of-magnitude larger than that of the aptamer alone(K_(D)≈0.29 μM) (Zimmermann et al, 2000). The observed increase in K_(D)is in agreement with the proposed mechanism (see below), where only theinactive conformation provides a formed aptamer that can bind ligand.Since shRNA switches can occupy both conformations, the apparentaffinity will be lower because ligand can only bind the inactiveconformation that is transiently present in a fraction of the shRNAswitch population.

The functionality of shRNA switches was assessed in mammalian cellculture. Applicants transiently transfected plasmids harboring S1 andvarious switch controls transcribed from a U6 promoter into HEK293Tcells stably expressing EGFP (Abbas-Terki et al, 2002). Flow cytometryanalysis revealed that S1 elicits intermediate knockdown of EGFP ascompared to the original shRNA targeting EGFP (sh) and a scrambled shRNA(neg) (FIGS. 1C & 1D), where the observed silencing by S1 can beattributed to activation of RNAi based on antisense inhibition of guidestrand activity (Hutvagner et al, 2004; Meister et al, 2004) (FIG. 8).In the presence of theophylline, GFP levels increased in adose-dependent manner for S1 but not for the control shRNAs. Theeffective concentration to achieve 50% activity (EC₅₀) for S1 of ˜300 μMwas much larger than the K_(D) of 5 μM measured in vitro, which can beprimarily attributed to a concentration drop in theophylline across thecellular membrane (Koch, 1956) (J Liang, J Michener, C Smolke,unpublished data, 2007). Mutating the aptamer core of S1 (S1′) greatlyreduced the observed theophylline dependence without perturbing basalexpression levels.

Taken together, S1 links theophylline concentration to GFP levels invivo through a relationship described by a component transfer function(FIG. 1D). Applicants obtained qualitatively similar results when shRNAswitches targeting EGFP were transiently transfected into other celllines (FIG. 9), suggesting that shRNA switches can be broadly applied indifferent cell lines and types.

Example 2 Mathematical Modeling Offers Tuning Parameters toPredicatively Modulate Component Transfer Functions

In this example, Applicants systematically evaluated the tuningcapabilities of shRNA switches with the aid of a mathematical modelrelating ligand concentration and target gene expression levels.Standard model parameters were incorporated to represent each chemicalstep from the proposed mechanism (Example 7). Applicants assumed the twoadopted conformations are at thermodynamic equilibrium, ligand onlybinds the inactive conformation, and the active conformation is solelyprocessed to a siRNA with a reduced efficiency as compared to theoriginal shRNA. These assumptions yield the following relationshipbetween relative expression levels of the target gene (f; output) andexogenous ligand concentration (L; input):

f=1−e·f _(shRNA[)1+K _(Comp)(1+K _(Apt) ·L)]^(−h),  (1)

where e is the processing efficiency, f_(shRNA) is the relativeknockdown achieved by the original shRNA (sh), K_(Comp) is thepartitioning coefficient between active and inactive conformations,K_(Apt) is the association constant for binding between ligand and theformed aptamer, and h is the hill coefficient to account fornonlinearity between siRNA concentration and target expression levels.

While complicated mathematical models have been developed for RNAi(Bartlett and Davis, 2006; Malphettes and Fussenegger, 2006; Raab andStephanopoulos, 2004), the approach utilizes a minimal parameter setthat is experimentally tractable, fully represents RNAi in the contextof shRNA switches, and captures the steady-state behavior of the system(FIG. 10). For one shRNA stem sequence and input ligand (fixedf_(shRNA), h), the model provides three tuning parameters that can bevaried to tune the component transfer function: K_(Comp), K_(Apt), and e(FIGS. 2A-2C). Varying K_(Comp) results in a concomitant and opposingvariation in EC₅₀ and basal expression levels, which are independentlytuned by K_(Apt) and e, respectively. In addition, as K_(Comp)approaches zero, basal expression levels approach a lower limit that isdependent on the value of e and is higher than that of the originalshRNA (FIG. 2D). Since each tuning parameter represents individual stepsin the proposed mechanism, Applicants examined how modifying thesequence in each domain, specifically the switching strand and aptamerdomains, corresponds to parameter variation in order to identify uniquetuning strategies (FIG. 3A).

Example 3 Switching Strand Tuning Strategies Enable PredictiveAlteration of the Component Transfer Function

Modifying switching strand base-pairing interactions is anticipated toreflect changes in K_(Comp), since this parameter represents thethermodynamic partitioning between active and inactive conformations.Applicants developed switching strand tuning strategies to targetmodifications to three regions within the switching strand domain: thelength of the switching strand on the 3′ end (FIGS. 3B & 3C) or the 5′end (FIGS. 3D & 3E), or the base-pairing complementarity (FIGS. 3F &3G). Applicants introduced iterative nucleotide changes under eachswitching strand tuning strategy and generated component transferfunctions as before. Regardless of the selected strategy, eachnucleotide change resulted in a shift in the response curve in line withthe model prediction for variation in K_(Comp). The results suggest thatdecreasing the extent of base-pairing interactions between the switchingstrand and the shRNA stem decreases the stability of or bias toward theinactive conformation (lower K_(Comp)), resulting in lower basalexpression levels and a higher EC₅₀. The trend towards higher EC₅₀ isconsistent with the order-of-magnitude difference between the apparentK_(D) of S1 observed in the in-line probing experiment and that reportedfor the aptamer alone (FIG. 1B). Thus, sequence modifications to theswitching strand that affect the extent of base-pairing solely map tovariation of K_(Comp).

Example 4 Aptamer Tuning Strategies Enable Predictive Alteration of theComponent Transfer Function

Although ligand binding to the formed aptamer directly relates toaptamer affinity, represented by K_(Apt), sequence changes in theaptamer domain may affect other parameters. To evaluate how sequencemodification of the aptamer domain corresponds to parameter variation,Applicants tested two theophylline aptamer variants (S11 and S12) withdissimilar K_(D) values (Zimmermann et al, 2000) and the mutated aptamer(S1′) (FIGS. 4A & 4B). Mutating the aptamer core (S1′) withoutperturbing shRNA switch secondary structure or sequence length resultedin a shift in EC₅₀, whereas decreasing aptamer affinity by decreasingthe aptamer stem length (S11 and S12) resulted in a shift in both EC₅₀and basal expression levels. The shifts in EC₅₀ for S11 and S12 matchedthe relative K_(D) measured in vitro for the aptamer variants alone(Zimmermann et al, 2000), suggesting that modulating aptamer affinity isreflected by variation in K_(Apt). However, K_(Apt) only affects EC₅₀,suggesting that either K_(Comp) or e varies with aptamer size. Since theswitching strand sequence is preserved for S1, S1′, S11, and S12,Applicants hypothesized that the shift in basal expression levelsindependent of K_(Apt) (most obvious in comparing the transfer functionsof S1 and S11) is solely attributed to the third tuning parameter e(FIG. 2C).

To evaluate the relationship between aptamer size and the tuningparameter e, Applicants replaced the theophylline aptamer with thesmaller xanthine aptamer (Kiga et al, 1998) or the larger tetracyclineaptamer (Berens et al, 2001). Since variation of e and K_(Comp) bothaffect basal expression levels, sole evaluation of e requires estimationof the lower limit of basal expression levels for vanishingly smallvalues of K_(Comp) (FIG. 2D). To this end, Applicants constructed atleast one shRNA switch with each aptamer that strongly prefers theactive conformation (low K_(Comp); see below) and measured GFP basalexpression levels of cells transfected with these constructs (FIG. 4C).Assay results indicated that aptamer size strongly correlated with thelower limit of basal expression levels. The results suggest that thetuning parameter e, which is predicted to have a significant effect onthe lower limit of basal expression levels, maps to the size of theaptamer domain.

These observations led to the specification of two general aptamertuning strategies: targeted changes in aptamer affinity without changingaptamer size alter K_(Apt), and targeted changes to aptamer size toalter the processing efficiency of the switch (e). Taken together,variation of K_(Apt) and e map to the aptamer domain and depend on thenature of the sequence modification.

Applicants also examined whether placement of new aptamers into theaptamer domain imparts new ligand dependence while preserving shRNAswitch functionality. Previous RNA-based regulatory platforms havedemonstrated alteration of ligand dependence by the modularincorporation of new aptamers (Bayer et al, 2005; Win et al, 2007) orminimal mutation of the base aptamer (Desai and Gallivan, 2004; Thompsonet al, 2002). Applicants evaluated the xanthine aptamer, since itproduced low basal expression levels and tightly binds the water solubleand non-cytotoxic small molecule hypoxanthine. Following construction ofshRNA switches that incorporate the xanthine aptamer by directreplacement of the aptamer domain, Applicants generated componenttransfer functions in HEK293T cells stably expressing EGFP. As observedfor S1, intermediate basal expression levels of GFP increased in adose-dependent manner that was abolished by mutating the aptamer core(FIGS. 4D & 4E). Contrary to model predictions, mutation of the xanthineaptamer increased the basal expression levels, which may be attributedto base pairing interactions with other shRNA switch domains. However,the shift in basal levels is less than that observed for changes in theswitching strand, suggesting that our model serves as a sufficient firstapproximation. Furthermore, the switching strand tuning strategies werepreserved as evidenced by the effect of changing the switching strandlength on the hypoxanthine response curves (FIGS. 4F & 4G). Thus, thesubject shRNA switch design can accommodate different aptamers to alterthe identity of the molecular input that regulates gene expression.

Example 5 Programming Transfer Functions by Combining Switching Strandor Aptamer Tuning Strategies

The ligand-regulated behavior of shRNA switches can be programmedthrough application of the switching strand and aptamer tuningstrategies described above. By combining these programming strategies, acollection of shRNA switches could be constructed that display finelytuned transfer functions and respond to a range of molecular inputs.

Based on the independence of the switching strand tuning strategies,Applicants first examined whether the strategies can be combined to finetune the component transfer function beyond the capabilities of anysingle strategy. To generate small deviations in the transfer functionof a parent shRNA switch, Applicants added compensatory nucleotidechanges under each switching strand tuning strategy in a step-wisemanner (FIGS. 5A & 5B): a point mutation (G68A) within the switchingstrand to increase complementarity, deletion of two base-pairs todecrease the switching strand length at the 5′ end, and a singleinsertion at the 3′ end to increase the switching strand length. Eachnucleotide change yielded the expected shift in the transfer functioncorresponding to the relative stabilization (increased K_(Comp)) ordestabilization (decreased K_(Comp)) of the inactive conformation. Thefinal switch, S10, displayed a transfer function slightly shifted fromthat of the parent switch, S4, demonstrating that nucleotide changesfollowing the three switching strand tuning strategies can be combinedto yield fine tuning of the component transfer function.

Example 6 An In Silico Framework Towards Component Sequence-to-TransferFunction Prediction

The construction of large-scale biological systems will require thesimultaneous optimization of the behavior of all system components toyield proper network behavior as suggested for natural (Suel et al,2007) and synthetic (Gardner et al, 2000; Yokobayashi et al, 2002)systems. While the transfer functions associated with shRNA switches andother synthetic riboswitches are amenable to physical tuning based onactual in vitro or in vivo experiments, a computational framework toeffectively navigate qualitatively functional sequences is necessary forthe rapid optimization of switch performance. Folding energetics dictateconformational partitioning and therefore switch performance for astrand displacement mechanism. RNA secondary structure predictionalgorithms, such as that described in Mathews et al (2004, incorporatedherein by reference) have the potential to perform accurate in silicoprediction of in vivo switch performance, although these algorithms havenot been sufficiently tested for in vivo folding dynamics. Toinvestigate the applicability of the secondary structure algorithms topredict in vivo switch behavior, Applicants sought to develop asequence-to-function relationship for switches using such algorithms incombination with our model (FIG. 6A). For convenience, shRNA is againused as an illustrative model, although the same principle and generalmethodology applies to other switch platforms (such as a ribozyme-basedswitch platform) as well.

Based on the tuning analysis above, Applicants identified K_(Comp) asthe sole parameter that reflects partitioning between active andinactive conformations and maps to the switching strand. The free energydifference (AG) between conformations is directly related to K_(Comp)such that transfer function prediction is possible by calculating ΔGfrom sequence information with the aid of structure predictionalgorithms, converting this value into K_(Comp), and inserting K_(comp)into equation (1) (see Example 2) to quantitatively relate ligandconcentration (L) and target gene expression levels (f). A fullydetermined model requires values for the remaining model parameters; asthese parameters are not currently amenable to calculation in silico,experimental estimation can be conducted with a minimal set ofexperiments based on our model construction. See Example 7 forderivation of the mathematical models.

Applicants first determined if ΔG values calculated from the algorithmcorrelate with the measured basal expression levels for shRNA switcheswith varying switching strand sequences. The implicit assumption is thatswitching strand alterations only affect conformational partitioning,which can be calculated with the structure prediction algorithms.Applicants evaluated ΔG (ΔG_(method)) by separating active and inactiveconformations based on the minimal free energy (MFE) and the weightedenergies from a partition function (PF) calculation (see below and FIG.12), where both methods are commonly used to evaluate RNA folding invitro and in vivo. These methods were employed to calculate ΔG_(method)for shRNA switches S1-10, which differ only in their switching strandsequence. To measure the correlation strength between ΔG_(method) andbasal expression levels for either method, Applicants performed aleast-squares fit using a three-parameter equation of the same form asour model with both data sets. Ideally, the fit relationship betweenΔG_(method) and measured basal expression levels should align with thesame relationship predicted by the model (FIG. 6B), where ΔG(ΔG_(model)) is related to K_(Comp) according to equation (3). However,both MFE and PF calculations failed to provide a significant correlationbetween ΔG_(method) and basal expression levels (FIG. 12), suggestingthat these methods are insufficient for accurate prediction of RNAfolding dynamics in vivo.

For all switching strand tuning strategies, increasing the stability ofthe inactive conformation always resulted in an increase in basalexpression (FIGS. 3B-3G). The MFE and PFE methods did not effectivelycapture each energetic shift potentially due to the inclusion of bindinginteractions outside of the major stems. Applicants hypothesized thatthe interactions outside of the switching strand domain are lessprevalent in vivo and are biasing the energetic calculations. To examinethis possibility, Applicants devised a third method, the stems method,that only accounts for the energetics of the major stem in eachconformation (FIG. 11). Implementing the stems method resulted in astrong correlation (R²=0.94) between basal expression levels andΔG_(method) (FIG. 6C).

Despite the absence of a perfect overlap between the stems methodcorrelation and that predicted by our model (FIGS. 6B & 6C), thecorrelation established a significant empirical link between shRNAswitch sequence and behavior in the absence of ligand. This correlationcan be assimilated into the model by equating basal expression levelspredicted by the fit equation and the model to determine therelationship between ΔG_(method) and K_(Comp). See Example 8 for freeenergy calculations and model extension.

Doing so yields a predictive component transfer function that is nowdependent on the calculated value of ΔG_(method):

$\begin{matrix}{{F_{model} = {1 - {e \cdot {f_{shRNA}\begin{bmatrix}{1 + \left\lbrack {\sqrt[h]{{\frac{e \cdot f_{shRNA}}{C_{1}}\left\lbrack {C_{2} + {\exp \left( {- \frac{\Delta \; G_{method}}{k_{B}N_{A}T}} \right)}} \right\rbrack}^{C_{3}}} - 1} \right\rbrack} \\\left( {1 + {K_{Apt} \cdot L}} \right)\end{bmatrix}}^{- h}}}},} & (2)\end{matrix}$

where C₁₋₃ are empirical constants from the fit correlation. Thisextended model provides a general framework for predicting shRNA switchtransfer functions from sequence information, where energetic valuesproduced from structure prediction algorithms are inserted into themodel for the prediction of switch behavior. While the extended modelcurrently requires parameter fitting to yield the predicted relationshipbetween ligand concentration and target gene expression levels, theframework establishes a starting point for the development of methodsthat rely on in silico calculations for transfer function predictionfrom sequence information.

Example 7 Derivation of Mathematical Model

Applicants developed a mathematical model to examine the mechanismthrough which shRNA switches mediate ligand control of RNA interference(RNAi). Instead of drawing from existing models (Bartlett and Davis,2006; Malphettes and Fussenegger, 2006; Raab and Stephanopoulos, 2004)that take into account the mechanistic steps and kinetics of RNAi thatare well characterized, Applicants chose to derive a simplified modelthat captures the steady-state behavior of shRNA switches and thefundamental mechanism that provides for ligand regulation of geneexpression. The goal was to develop a model that predicts the relativesteady-state expression levels of the target gene (f; output) as afunction of exogenous ligand concentration (L; input), and can be easilyadapted to predict shRNA switch activity in different cellularenvironments.

To accomplish this, Applicants began with the proposed mechanism forshRNA switch functionality (FIG. 1A). This mechanism asserts that asingle shRNA switch can adopt two conformations due to distinctbase-pairing interactions. The active conformation (left) is processedby the RNAi machinery to an siRNA that initiates RNAi-mediated silencingof target transcripts. Processing includes nuclear export by Exportin-5(Yi et al, 2003) and cleavage by the RNase III-like enzyme Dicer(Ketting et al, 2001). Conversely, the inactive conformation (middle) isnot processed by the RNAi machinery. Ligand binding to the formedaptamer domain in the inactive conformation stabilizes this conformation(right), thereby reducing overall processing of the shRNA switch to ansiRNA.

Model derivation began by assuming that the three conformations (active,inactive, and inactive bound to ligand) are at thermodynamic equilibriumas determined by K_(Comp) and K_(Apt). K_(Comp) is the equilibriumpartitioning coefficient between active and inactive conformations,while K_(Apt) is the association constant for binding between ligand andthe inactive conformation. When normalized to the total shRNA switchconcentration, the fraction of shRNA switches in the active conformationis:

$\begin{matrix}{{{\lbrack\rbrack}} = {\frac{1}{1 + {K_{comp}\left( {1 + {K_{apt} \cdot L}} \right)}}.}} & (1)\end{matrix}$

The next step was correlating the fraction of shRNA switches in theactive conformation to relative expression levels of the target gene.Previous models have highlighted the importance of absolute expressionlevels of the RNAi substrate, target gene transcripts, and theRNA-induced silencing complex (RISC), as well as the rate of celldivision (Bartlett et al, 2006). Recent work has elaborated on themechanism of RNAi, including the emerging role of Dicer binding partnersTRBP and PACT (Gregory et al, 2005; Kok et al, 2007; Lee et al, 2006),association of RISC and Dicer (Gregory et al, 2005), shuttling of thecleaved siRNA from Dicer to RISC (Gregory et al, 2005), cleavage andrelease of the passenger strand (Matranga et al, 2005; Rand et al,2005), target site availability for efficient degradation of the targettranscript (Westerhout and Berkhout, 2007), and the potential forsaturation of Exportin-5 (Grimm et al, 2006; Yi et al, 2005). Ratherthan offer a descriptive model of RNAi that incorporates all of thesemechanisms that are still under investigation, we chose an empiricalroute that requires minimal experimental data.

Excluding nuclear export by Exportin-5, the mechanistic steps describedabove apply to the linear cascade downstream of and including Dicerrecognition and processing. Incorporation of three parameters,f_(shRNA), e, and h, can account for the dynamics of these steps.f_(shRNA) is the relative knockdown achieved by the original shRNA—anRNA molecule comprised of a loop region and the shRNA stem sequence, eis the efficiency of shRNA switch processing by the RNAi machinery, andh is the hill coefficient that accounts for the nonlinearity between theconcentration of Dicer-cleaved siRNAs and relative expression levels ofthe target gene. To capture the correlation between the prevalence ofthe active conformation and target gene expression levels, we used thefollowing relationship:

Introducing equation (1) into equation (2) yields the final form of themodel:

f=1−e·f _(shRNA[)1+K _(Comp)(1+K _(Apt) ·L)]^(−h),  (3)

where the relative expression levels of the target gene (f) are afunction of exogenous ligand concentration (L).

The power of this model lies in the ability to calculate realisticparameter values from a minimal set of experiments: f_(shRNA) can befound in one experiment by measuring the relative knockdown of thetarget gene induced by an shRNA that contributes the shRNA stem, e canbe calculated from basal expression levels from a few shRNA switchesthat strongly prefer the active conformation, and h can be calculated bygenerating a ligand response curve with one shRNA switch—as long asadministration of the highest ligand concentration results in negligibleknockdown of the target gene. The remaining model parameters, K_(Comp)and K_(Apt), can be found from the same response curve used to calculatee, since varying K_(Apt) only changes the EC₅₀ while varying K_(Comp)changes both EC₅₀ and basal expression levels. A summary of the modelparameters and how values are experimentally obtained are included inTable III below.

TABLE III Description of model parameters parameter initialdetermination description K_(Comp) fit to data Equilibrium constantbetween active and inactive conformations (—) K_(Apt) fit to dataAssociation constant between ligand and formed aptamer (1/μM) eextrapolated from data RNAi processing efficiency (—) f_(shRNA) fromshRNA data Relative knockdown by original shRNA (—) h fit to data Hillcoefficient (—)

To investigate the validity of the model, Applicants experimentallydetermined model parameter values as described above: f_(shRNA) wasequated to the knockdown achieved with the original shRNA targeting EGFP(sh); e was calculated from the average basal expression levels producedby shRNA switches S5, S7, S9, and S10; and K_(Comp), K_(Apt), and h weredetermined by a model fit of the theophylline response curve for S1. Theresulting parameter values are shown in FIG. 10. The fit curve alignswith the response curve for S1, and the fit parameter values arerealistic as described below for K_(Apt) and K_(Comp). The EC₅₀ isrelated to K_(Apt) and K_(Comp) according to the following:

EC ₅₀=(1+K _(Comp) ⁻¹)K _(Apt) ⁻¹,  (4)

From the in-line assay results, the ratio of the apparent K_(D) of S4t(5 μM) to the K_(D) of the aptamer alone (0.29 μM (Zimmermann et al,2000)) was ˜17. Solving for K_(Comp) in equation (4) yields a value of0.06. While this is below the fit value from the S1 data of 0.17, S1 hasone less base pair than S4 contributed by the competing strand. Thus,the value from S4t is anticipated to be closer to 0.17 if the extra basepair is included. The fit value for K_(Apt) (0.016 μM⁻¹) from the S1data was lower than that for the aptamer alone (3.4 μM), which can beattributed to a theophylline concentration drop across the cellularmembrane as observed in E. coli (Koch, 1956) and S. cerevisiae (J Liang,J Michener, C Smolke, unpublished data, 2007). Hence the modelfaithfully follows the underlying mechanism of ligand regulation of geneexpression mediated by shRNA switches and can capture in vivo behaviorby utilizing a minimal set of experiments.

References cited in this example are listed herein below.

-   Bartlett D W, Davis M E (2006) Insights into the kinetics of    siRNA-mediated gene silencing from live-cell and live-animal    bioluminescent imaging. Nucleic Acids Res 34: 322-333.-   Gregory R I, Chendrimada T P, Cooch N, Shiekhattar R (2005) Human    RISC couples microRNA biogenesis and posttranscriptional gene    silencing. Cell 123: 631-640.-   Grimm D, Streetz K L, Jopling C L, Storm T A, Pandey K, Davis C R,    Marion P, Salazar F, Kay M A (2006) Fatality in mice due to    oversaturation of cellular microRNA/short hairpin RNA pathways.    Nature 441: 537-541.-   Ketting R F, Fischer S E, Bernstein E, Sijen T, Hannon G J, Plasterk    R H (2001) Dicer functions in RNA interference and in synthesis of    small RNA involved in developmental timing in C. elegans. Genes Dev    15: 2654-2659.-   Koch A L (1956) The metabolism of methylpurines by Escherichia    coli. I. Tracer studies. J Biol Chem 219: 181-188.-   Kok K H, Ng M H, Ching Y P, Jin D Y (2007) Human TRBP and PACT    directly interact with each other and associate with dicer to    facilitate the production of small interfering RNA. J Biol Chem 282:    17649-17657.-   Lee Y, Hur I, Park S Y, Kim Y K, Suh M R, Kim V N (2006) The role of    PACT in the RNA silencing pathway. EMBO J 25: 522-532.-   Malphettes L, Fussenegger M (2006) Impact of RNA interference on    gene networks. Metab Eng 8: 672-683.-   Matranga C, Tomari Y, Shin C, Bartel D P, Zamore P D (2005)    Passenger-strand cleavage facilitates assembly of siRNA into    Ago2-containing RNAi enzyme complexes. Cell 123: 607-620.-   Raab R M, Stephanopoulos G (2004) Dynamics of gene silencing by RNA    interference. Biotechnol Bioeng 88: 121-132.-   Rand T A, Petersen S, Du F, Wang X (2005) Argonaute2 cleaves the    anti-guide strand of siRNA during RISC activation. Cell 123:    621-629.-   Westerhout E M, Berkhout B (2007) A systematic analysis of the    effect of target RNA structure on RNA interference. Nucleic Acids    Res.-   Yi R, Doehle B P, Qin Y, Macara I G, Cullen B R (2005)    Overexpression of exportin 5 enhances RNA interference mediated by    short hairpin RNAs and microRNAs. RNA 11: 220-226.-   Yi R, Qin Y, Macara I G, Cullen B R (2003) Exportin-5 mediates the    nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev    17: 3011-3016.-   Zimmermann G R, Wick C L, Shields T P, Jenison R D, Pardi A (2000)    Molecular interactions and metal binding in the theophylline-binding    core of an RNA aptamer. RNA 6: 659-667.

Example 8 Free Energy Calculations and Model Extension

The model derived in Example 7 identified different tuning trends thatwere observed in our experimental analysis, although this form of themodel only predicts qualitative shifts in the transfer function basedupon nucleotide changes to a parent shRNA switch. We sought to augmentthe model with predictive capabilities for the forward design of shRNAswitch sequences that yield desired transfer functions. We initiallyfocused on K_(Comp), the partitioning coefficient between active andinactive conformations, since it solely captured the effect of multiplechanges to the switching strand and has a thermodynamic basis. Underbasic thermodynamic assumptions, K_(Comp) is related to the free energydifference (AG) between the active and inactive conformations accordingto

$\begin{matrix}{{{\Delta \; G} = {{E()} = {{- N_{A}}k_{B}{T \cdot {\ln \left( K_{Comp} \right)}}}}},} & (1)\end{matrix}$

where N_(A) is Avogadro's number, k_(B) is the Boltzmann constant, and Tis temperature (K). If ΔG can be calculated for a given shRNA switchsequence, then the corresponding value of K_(Comp) can be calculated.When paired with the other experimentally-determined parameter values(Example 7), this value of K_(Comp) can then be used in the model topredict the transfer function relating ligand concentration and relativegene expression levels. The initial challenge is calculating anexperimentally valid ΔG from a given shRNA switch sequence.

Free Energy Calculation

To calculate AG, we employed the RNA secondary structure predictionprogram RNAStructure 4.5 (Mathews et al, 2004) to output structural andenergetic information for a given sequence. The program's dynamicfolding algorithm utilizes empirical energy values measured in vitro(Mathews et al, Proc Natl Acad Sci USA 101: 7287-7292, 2004) to predictRNA conformations and their relative free energy. Since application ofthe program to in vivo folding has rarely been addressed (Mathews et al,2004), we first asked if ΔG values calculated from the program(ΔG_(method)) correlated with measured basal expression levels for eachshRNA switch. Two commonly used methods were initially employed tocalculate ΔG_(method) for S1-10 (switches with the same aptamer domainand shRNA stem): minimal free energy of the active and inactiveconformation (MFE method) and partition function calculation to find therelative probability of either general conformation (PF method).ΔG_(method) values were then plotted with the associated basalexpression levels measured in vivo (Table II) and compared to theexpected trend from the model (ΔG_(model); FIG. 6B). A three-parameterequation with the same mathematical form as the model was then fit toeach data set using a least-squares analysis to evaluate the correlationstrength, since a strong correlation is necessary for accurateprediction of the transfer function. The mathematical form used to fitthe data was:

$\begin{matrix}{{f_{fit} = {1 - {C_{1}\left\lbrack {C_{2} + {\exp \left( {- \frac{\Delta \; G_{method}}{k_{B}N_{A}T}} \right)}} \right\rbrack}^{- C_{3}}}},} & (2)\end{matrix}$

where C1, C2, and C3 are fit constants and f_(fit) is the basalexpression of the target gene for the fit curve.

MFE Method

The minimal free energy conformation—the most stable conformation—hasbeen considered to be representative of the actual tertiary structure,and the free energy of this conformation is often considered torepresent overall energetics of the RNA sequence. Under the MFE method,the free energy is recorded for the most stable active and inactiveconformation. The difference in these free energy values is thenreported as ΔG_(method). The resulting plot (FIG. 12) shows nosignificant correlation and an associated weak fit (R²=0.35), suggestingthat this method is insufficient for predicting transfer functions.

PF Method

Calculation of the partition function is a more advanced and consideredto be a more accurate method for the approximation of RNA energetics.All possible secondary structure conformations and their energies arecalculated in order to identify the most prevalent conformation, whichoften deviates from the minimal free energy conformation. Under the PFmethod, the program outputs the probability of a given base-pair basedon the partition function calculation. To convert these probabilitiesinto a value of ΔG, we first found the smaller value of the base-pairprobabilities near the top and bottom of the upper shRNA stem (startingat the stem bulge) in the active conformation and the stem formed by theswitching strand and the shRNA stem in the inactive conformation (FIG.11). Base-pairs were chosen such that the same nucleotide in the shRNAstem was part of the selected base-pair in both conformations. Thisensures that a base-pair probability only applies to one of the twoconformations. In other words the sum of the base-pair probabilitiesthat include the same nucleotide for both conformations should always beless than one. Ideally, the sum should equal one, where all calculatedsums for S1-10 were between 85% and 99% (data not shown). The value ofΔG_(method) can be calculated from the base-pair probabilities accordingto the following:

$\begin{matrix}{{{\Delta \; G_{method}} = {{- k_{B}}N_{A}{T \cdot {\ln \left( \frac{P_{I}}{P_{A}} \right)}}}},} & (3)\end{matrix}$

where P_(A) and P₁ are the base-pair probabilities representing theactive or inactive conformations, respectively. ΔG_(method) values werecalculated using the PF method and plotted in the same way as above(FIG. 12). The PF method provided a better fit (R²=0.53) when comparedto the MFE method that qualitatively matched the model trend, althoughthe fit is not suitable for predictive purposes either.

Stems Method

While increasing the extent of base-pairing between the switching strandand shRNA stem always resulted in an increase in basal expression levels(FIG. 3B-G), the MFE and PF calculations output predicted an increase ordecrease in free energy changes based on binding interactions outside ofthe major stems. We attributed the inaccuracy of the MFE and PF methodsto the equal weight placed on these binding interactions. To removethese contributions to the energetic calculation, we devised a thirdmethod we term the stems method. This method only accounts for theenergetic contributions from the major stems in the active and inactiveconformations. The major stem for the active conformation spans from theshRNA stem bulge to the top of the shRNA stem, while the major stem forthe inactive conformation includes base-pairs formed between the shRNAstem and the switching strand (FIG. 11). The lower portion of the shRNAstem is ignored since it is present in both conformations. As before, wecalculated ΔG_(method) for S1-10 and plotted these values against thebasal expression levels. The resulting plot (FIG. 6C) shows a strongcorrelation (R₂=0.94), a significant improvement over the other methods.

It is surprising yet insightful that the most accurate method onlyaccounts for energetic contributions from regions that interact with theswitching strand, which is precisely and solely where K_(Comp) maps. Aninequality does exist between the fit curve from the stems method andmodel predictions in terms of the abscissa values and curve slope, whichsuggests that sequences outside of the major stem contribute to foldingenergetics in vivo in a way that is improperly treated by the MFE or PFmethod.

Model Extension

Based on the strong correlation between ΔG_(method) calculated fromshRNA switch sequence and in vivo basal expression levels, the fit curvefrom the stems method (but not the MFE or PF methods) can beincorporated into our model for the forward design of shRNA switches.This is accomplished by converting the value of ΔG_(method) calculatedfrom the stems method into K_(Comp) that can be used in the model topredict the transfer function. To perform this conversion, f from themodel equation and f_(fit) from the curve fit are set equal to eachother. For successful conversion, the dynamic range (the range of f) ofthe model and fit curves must match exactly. This can be done byensuring that

1−e·f _(shRNA) =f _(fit)(ΔG _(method)→∞),  (4)

where e and f_(shRNA) are model parameters. Once set equal to eachother, K_(Comp) can be found in terms of ΔG_(method):

$\begin{matrix}{K_{Comp} = {\sqrt[h]{{\frac{e \cdot f_{shRNA}}{C_{1}}\left\lbrack {C_{2} + {\exp \left( {- \frac{\Delta \; G_{method}}{k_{B}N_{A}T}} \right)}} \right\rbrack}^{C_{3}}} - 1.}} & (5)\end{matrix}$

Replacing K_(Comp) in the model with equation (5) yields the extendedmodel:

$\begin{matrix}{f_{model} = {1 - {e \cdot {{f_{shRNA}\begin{bmatrix}{1 + \left\lbrack {\sqrt[h]{{\frac{e \cdot f_{shRNA}}{C_{1}}\left\lbrack {C_{2} + {\exp \left( {- \frac{\Delta \; G_{method}}{k_{B}N_{A}T}} \right)}} \right\rbrack}^{C_{3}}} - 1} \right\rbrack} \\\left( {1 + {K_{Apt} \cdot L}} \right)\end{bmatrix}}^{- h}.}}}} & (6)\end{matrix}$

Following experimental determination of the remaining model parametervalues (Example 7), this equation can be used to predict relativeexpression levels of the target gene (f_(model)) as a function of ligandconcentration (L) by calculating ΔG_(method) under the stems methodusing RNAStructure.

Example 9 Model-Guided Forward Design of shRNA Switches with OptimizedTransfer Functions

To apply the extended model to the forward design of shRNA switches withdefined functional properties, Applicants sought to design atheophylline-regulated shRNA switch displaying a maximized dynamic range(η). Here, η is defined as the ratio of GFP levels at high (3 mM) andlow (1 μM) theophylline concentrations. Applicants used the extendedmodel to calculate the range of ΔG values where η is maximized. Modelpredictions suggest that η is maximized for switches with ΔG_(method)˜−3kcal/mol and that use of the smaller theophylline aptamer (higher e)yields a higher maximum (FIG. 6D). To evaluate the predicted landscape,Applicants designed new shRNA switches (S13-25) that include the smallertheophylline aptamer and display ranging ΔG values, generated componenttransfer functions, and calculated η. Plotting ΔG_(method) against themeasured value of η for all theophylline-regulated shRNA switches(S1-25; FIG. 6E) shows a maximum for switches containing the smallertheophylline aptamer that is higher than that for the switchescontaining the larger aptamer. Furthermore, both maxima existed atΔG_(method)˜3 kcal/mol as predicted by the extended model supplementedwith the empirical parameter values, and the best switch (S13) wasapproximately equal to the theoretical maximum of η according to modelpredictions (η_(max,theor)˜5). Flow cytometry data illustrate theimprovement in dynamic range (FIG. 13) for the best shRNA switch (S13;FIG. 6G) as compared to the original shRNA switch (S1; FIG. 6F).

To examine the generality of shRNA switch design and functionality,Applicants designed a set of shRNA switches targeting the endogenous Laprotein. Following selection of an shRNA sequence that yields moderateknockdown of La as ascertained by qRT-PCR, various switching strandsequences covering a range of ΔG values were combined with the smallertheophylline aptamer to yield six shRNA switches (L1-6). Each shRNAswitch showed variable response to 1.5 mM theophylline that was notobserved for the base shRNA (FIG. 14). As observed for the GFP-targetingshRNA switches, use of the stems method provided a suitable correlationbetween basal levels and ΔG_(method). Supplying the model with fitvalues for C₁₋₃ yielded a predicted dynamic range trend that closelymatched the experimental data. Interestingly, when the values off_(shRNA) and e calculated from the base shRNA and an shRNA switchpreferentially adopting the active conformation (L6) were combined withthe remaining parameter values from the GFP experiments, the resultingtrend predicted the same maximal dynamic range with a shifted value ofΔG_(method) that maximizes dynamic range. This suggests thatsequence-specific factors affect calculations with the stems method suchthat empirical values are specific to individual sequences andexperimental conditions. However the stems method produced a strongcorrelation such that the model may be implemented in future designs bygenerating a small set of shRNA switches covering ΔG_(method) values ofapproximately −5 to 0 kcal/mol and measuring basal expression levels.Thus, shRNA switches can be constructed to target different genes andthe model can be used as a tool for forward design.

A comparison between the framework described here and a recentlydescribed ligand-controlled shRNA system (An et al, 2006) highlightsimportant design strategies to engineer domain swapping and tuning ofthe transfer function into synthetic riboswitch systems. In the previousdesign, ligand control of RNAi was achieved through direct coupling ofthe theophylline aptamer and an shRNA stem. This design inherentlylimits aptamer swapping since the aptamer must perform ligand bindingcoordinated with modulation of Dicer processing, and prevents tuning ofthe transfer function since sequence changes that modulate Dicerprocessing cannot be implemented without a complete loss of ligandresponsiveness. In contrast, the framework described herein is based onthe coupling of three distinct domains that carry out separate functionsnecessary to convert ligand binding into modulation of RNAi activity.This system requires that the aptamer performs one function—ligandbinding—and the modulation of RNAi processing is performed by a separatedomain, the switching strand. The switching strand permits fine tuningof the transfer function and enables modular coupling of the aptamer andshRNA stem domains, as confirmed by independently replacing each domainand demonstrating preservation of functionality.

Applicants developed a model to enhance the understanding of shRNAswitch activity and identified five tuning strategies reflected in threemodel parameters, K_(Comp), K_(Apt), and e that map specifically tosequence changes in the switching strand or aptamer domains. This modelalso established important shRNA switch design guidelines. The first isthat basal expression levels are determined by a collection of factors:shRNA potency (f_(shRNA)), shRNA switch processability (e), andprevalence of the active conformation (K_(Comp)). To achieve a desiredbasal expression level, all factors must be considered in the switchdesign. Another guideline originates from the observation that largeraptamers coincided with increased basal expression levels, potentiallydue to sterically hindering processing by the RNAi machinery. Thespecific contribution of secondary or tertiary structure to theinhibitory effect is unclear, although further understanding of how theRNAi machinery specifically interacts with the shRNA throughcrystallographic or mutational studies may shed light on thisdependence. Our results suggest that shRNA switch sequence length has anupper limit before compromising activity, where future engineeringefforts may focus on alleviating or entirely removing this limitation.Furthermore, if achieving low basal expression levels is critical and aset of aptamers against the same ligand are available, use of smalleraptamers may be preferred even at a cost to aptamer affinity. Such aguideline may even direct library design for the selection of newaptamers by placing an upper limit on the length of the randomizedsequence.

Applicants also incorporated RNA folding algorithms into our model forin silico prediction of shRNA switch behavior in vivo. The resultingmodel yielded a framework for the forward design of shRNA switches withspecified functional properties. This was achieved by linking RNAsecondary structure prediction algorithms, which convert sequenceinformation into energetic values, to our model, which convertsenergetic values into switch behavior to provide an empiricalsequence-function relationship. The specific method used to calculatethe free energy difference (ΔG_(method)) between active and inactiveconformations deviated from commonly used methods (MFE and PFcalculations) based on observations from the experimental tuning trends.Our alternative method may provide a better correlation withexperimental results by focusing the prediction of K_(Comp) to theregion of the switch in which the switching strand binding events areoccurring, ignoring energetic contributions by other regions of theswitch molecule that may not be relevant to the in vivo conformationalswitching process. Our analysis moves towards directsequence-to-function relationships and suggests that commonly usedmethods for predicting RNA structure and behavior should be carefullyevaluated when applied to in vivo environments. RNA folding in vivo is acomplex process, and algorithms that account for folding kinetics(Danilova et al, 2006) and ulterior structural formation (Parisien andMajor, 2008), such as pseudoknots or non-canonical base-pairinginteractions, may increase the accuracy of the model as well as provideinsight into sequences that deviate from model predictions (FIG. 6E; andFIG. 10). Other algorithms may also be used. Such algorithms may providethe ability to rapidly scan suboptimal structures, to calculate theenergetics of multiple RNA strands, and to perform a partition functioncalculation, etc. While the PF method did not produce a strongcorrelation using existing algorithms, it may be useful for the subjectmethod using an algorithm designed to account for non-canonicalbase-pairing interactions.

Based on the demonstrated modularity and tunability of our platform,shRNA switches can be implemented towards various applications. As onepotential application, shRNA switches could be applied to diseasetherapy by sensing intracellular disease markers and inducing apoptosisor cell cycle arrest only in the affected cells. When acontext-dependent concentration threshold divides diseased and normalcells, tunability is essential to reduce the likelihood of falsepositives or negatives. As long as the sensitivity of the threshold doesnot exceed the dynamic range of the shRNA switch, the response curve canbe finely tuned to ensure that basal and ligand-saturating levelscoincide with survival or the induction of apoptosis. In addition, shRNAswitches could be integrated into synthetic genetic circuits to generateadvanced control schemes in biological systems. Such systems oftenexhibit complex dependencies on the dynamics of component interactions,and tuning of component behavior is often necessary to achieve optimalsystem performance. Through the fine tuning strategies and model-guidedforward design tools described here, shRNA switches may be used toaddress challenges faced in biological network design and serve ascomplex regulatory components in synthetic biology.

The following section provides exemplary materials and methods used inExample 1-9, which are for illustrative purpose only, and are notnecessarily limiting in any respect.

Materials and Methods

Plasmid construction. All shRNAs were cloned into pSilencer 2.1-U6 puro(Ambion). The original shRNA present in pSilencer was used as ascrambled shRNA control. The pSilencer backbone was modified toco-express DsRed-Express in 293T cells by cloning the SV40 origin ofreplication, CMV IE promoter, and DsRed-Express into the NsiI/MfeIrestriction sites. The original XhoI site present in the backbone wasalso removed by XhoI cleavage, extension with the large Klenow fragment(New England Biolabs), and ligation. To clone the shRNA switches, theoriginal shRNA followed by a 6-nucleotide (nt) string of T's was clonedinto BamHI/HindIII directly downstream of the U6 promoter. The originalshRNA was converted into an shRNA switch by cloning the remainingsequence into XhoI/XbaI contained within the shRNA loop region. Allcloning steps involved annealing of 5′-phosphorylated syntheticoligonucleotides (Integrated DNA Technologies) and ligation into thebackbone vector. All restriction enzymes and T4 DNA ligase werepurchased from NEB. All constructs were sequence-verified (Laragen,Inc.), where sequences are provided in Table I.

TABLE I Cloning sites Name Aptamer Sequence (5′/3′) Database # neg N/AGGATCCACTACCGTTGTTATAGGTGTTCAAGAGACA BamHI/ pCS628CCTATAACAACGGTAGTTTTTTGGAAAAGCTT HindIII shGGATCCGGTGCAGATGAACTTCAGGGTCAGCTCGA pCS741GTCTAGAGCTGACCCTGAATCATCTGCACCTTTTTT GGAAGCTT shLGGATCCGGCTTCCCAACGATGATGCAACTCCTCGA pCS1457GTCTAGAGGAGTTGCATCAGTTGGGAAGCCTTTTTT GGAAGCTT S1 theophylineCTCGAGATACCAGCATCGACTCTTCGATGCCCTTCG Xhol/Xbal pCS630GCAGCTCGGGCTGACCCTGACTAGA S1′ CTCGAGGACCCAGCATCGACTCTTCGATGAAATG pCS847GCAGCTCGGGCTGACCCTGACTAGA S2 CTCGAGATACCAGCATCGACTCTTCGATGCCCTTG pCS633GCAGCTCGGGCTGACCCTGAAGCTAGA S3 CTCGAGATACCAGCATCGACTCTTCGATGCCCTTGpCS631 GCAGCTCGGGCTGACCCTGAACTAGA S4 CTCGAGATACCAGCATCGACTCTTCGATGCCCTTGpCs628 GCAGCTCGGGCTGACCCTGCTAGA S5 CTCGAGATACCAGCATCGACTCTTCGATGCCCTTGpCS632 GCAGCTCGGGCTGACCCTCTAGA S6 CTCGACGATACCAGCATCGACTCTTCGATGCCCTTpCS848 GGCAGCGTCGGGCTGACCCTGCTAGA S7CTCGATACCAGCATCGACTCTTCGATGCCCTTGGCA pCS807 GCGAGCTGACCCTGCTAGA S8CTCGAGATACCAGCATCGACTCTTCGATGCCCTTG pGS629 GCAGCTCGAGCTGACCCTGCTAGA S9CTCGAGATACCAGCATCGACTCTTCGATGCCCTTG pCS1005 GCAGCTCGAGCTGATCCTGCTAGA S10CTCGATACCAGCATCGACTCTTCGATGCCCTTGGCA pCS808 GCGAGCTGACCCTGACTAGA S11CTCGAGATACCAGCCGAAAGGCCCTTGGCAGCTCG pCS634 GGCTGACCCTGACTAGA S12CTCGAGATACCACCGAAAGGCCTTGGCAGCTCGGG pCS635 CTGACCCTGACTAGA S13CTCGAGATACCAGCCGAAAGGCCCTTGGCAGCTCG pCS911 GGCTGACCCTGCTAGA S14CTCGATACCAGCCGAAAGGCCCTTGGCAGCGAGCT pCS908 GACCCTGCTAGA S15CTCGATACCAGCCGAAAGGCCCTTGGCAGCGGGCT pC5909 GACCCTGCTAGA S16CTCGATACCAGCCGAAAGGCCCTTGGCAGCGAGCT pCS910 GACCCTGACTAGA S17CTCGAGATACCAGCCGAAAGGCCCTTGGCAGCTCG pCS941 AGCTGACCCTGCTAGA S18CTCGAGATACCAGCCGAAAGGCCCTTGGCAGCTCG pCS942 AGCTGACCCTACTAGA S19CTCGAGATACCAGCCGAAAGGCCCTTGGCAGCTCG pCS1001 GGCTGACCCTGAACTAGA S20CTCGAGATACCAGCCGAAAGGCCCTTGGCAGCTCG pCS1002 GGCTGACCCTGAAGCTAGA S21CTCGAGATACCAGCCGAAAGGCCCTTGGCAGCTCG pCS1003 GGCTGACCCTGGCTAGA S22CTCGATACCAGCCGAAAGGCCCTTGGCAGCGAGCT pCS1004 GACCCTGAACTAGA S23CTCGAGATACCAGCCGAAAGGCCCTTGGCAGCTCG pCS1061 AGCTGATCCTGCTAGA S24CTCGAGATACCAGCCGAAAGGCCCTTGGCAGCTCG PCS1062 GGCTGATCCTGCTAGA S25CTCGAGATACCAGCCGAAAGGCCCTTGGCAGCTCG pCS1063 GGCTGATCCTGACTAGA X1xanthine/ CTCGAGTGTATTACCCAGCGAGGTCGACTCGAGCT Xhol/ pCS870 guanineGACCCTGACTAGA Xbal X1′ CTCGAGTTTCAAACCCAGCGAGGTACACTCGAGCT pCs913GACCCTGACTAGA X2 CTCGAGTGTATTACCCAGCGAGGTCGACTCGAGCT pCS972GACCCTGAACTAGA X3 CTCGAGTGTATTACCCAGCGAGGTCGACTCGAGCT pGS869GACCCTGCTAGA T1 tetracyline CTCGAAAACATACCAGAGAAATCTGGAGAGGTGAA Xhol/pCS893 GAATACGACCACCTCGAGCTGACCCTGACTAGA Xbal T2CTCGAAAACATACCAGAGAAATCTGGAGAGGTGAA pCS894GAATACGACCAGCTCGAGCTGACCCTGAACTAGA T3CTCGAAAACATACCAGAGAAATCTGGAGAGGTGAA pCS895GAATACGACCACCTCGAGCTGACCCTGCTAGA L1 theophylineCTCGAGATACCAGCCGAAAGGCCCTTGGCAGCTCG Xhol/ pCS1458 AGGAGTTGCATCCTAGA XbalL2 CTCGAGATACCAGCCGAAAGGCCCTTGGCAGCTCG pCS1459 AGGAGTTGCATTCTAGA L3CTCGAGATACCAGCCGAAAGGCCCTTGGCAGCTTG pCS1460 AGGAGTTGCATCCTAGA L4CTCGAGATACCAGCCGAAAGGCCCTTGGCAGCTTG pCS1462 AGGAGTTGCATACTAGA L5CTCGAGATACCAGCCGAAAGGCCCTTGGCAGCTCG pCS1463 AGGAGTTGCACTAGA L6CTCGAGATACCAGCCGAAAGGCCCTTGGCAGCTTG pCS1464 AGGAGTTGCACTAGA

TABLE II Basal expression ΔG (method) Name Aptamer levels (%) η MFE PFStems neg N/A 90.7 1.07 N/A sh 4.6 1.19 N/A S1 theophylline 33.8 2.71−0.1 0.6 −5.1 S1′ 37.0 1.39 −0.3 0.8 −5.1 S2 76.7 1.25 0.0 −0.2 −8.6 S352.8 1.68 −0.6 −0.1 −6.1 S4 23.8 3.65 3.2 3.4 −3.2 S5 21.1 3.58 6.2 6.2−0.6 S6 42.5 2.20 2.1 2.7 −5.7 S7 14.5 2.32 2.5 3.2 0.4 S8 37.3 2.47 0.31.2 −4.1 S9 12.1 4.75 2.9 3.2 −1.5 S10 29.4 3.60 −0.9 −0.1 −1.5 S11 20.04.20 0.6 0.9 −5.1 S12 13.4 4.06 −0.1 0.8 −5.1 S13 16.7 5.61 4.0 3.8 −3.2S14 14.3 2.86 2.6 3.3 0.4 S15 11.2 2.56 9.9 9.1 1.3 S16 21.7 3.85 0.10.1 −1.5 S17 17.5 3.12 2.5 2.0 −4.1 S18 19.5 3.89 5.7 4.5 −1.5 S19 38.32.38 −0.6 0.0 −6.1 S20 49.8 1.75 0.0 −0.2 −8.6 S21 10.8 2.52 4.6 4.6−4.2 S22 35.0 2.73 −0.9 −0.9 −2.4 S23 16.6 3.30 5.1 4.4 −1.5 S24 10.83.00 6.6 6.1 −0.6 S25 11.8 2.63 3.2 3.3 −2.5

Preparation of RNAs. S4t was transcribed in vitro from an annealedtemplate containing the T7 promoter (5′-TTCTAATACGACTCACTATAGGG-3′,where G is the first transcribed nucleotide) using the Ampliscribe T7transcription kit (Epicentre) according to the manufacturer'sinstructions. Following transcription and DNase treatment,unincorporated NTPs were removed using a NucAway clean-up column(Ambion). The 5′ phosphates were subsequently removed using Antarcticphosphatase (NEB). Dephosphorylated RNA was then gel-purified on a 6%denaturing polyacrylamide gel and quantified using a ND-1000spectrophotometer (Nanoprop). RNAs were 5′ radiolabeled using T4 PNK(NEB) and [γ-³²P]-ATP, purified using a NucAway clean-up column, andgel-extracted on a 6% denaturing polyacrylamide gel.

In-line probing. In-line probing was conducted as described previously(Soukup et al, 1999). After heating at 70° C. for 2 min followed by slowcooling to room temperature, 5′ radiolabeled RNAs (0.2 μmol) wereincubated for 40 hours at 25° C. in varying amounts of theophylline with50 mM Tris-HCl pH 8.5, 20 mM MgCl₂. Reactions were terminated by addingan equal volume of loading buffer (10 M urea, 1.5 mM EDTA). The alkalinehydrolysis ladder was generated by incubating RNA in 50 mM NaHCO₃/Na₂CO₃pH 9.2, 1 mM EDTA for 6 min at 95° C. The G-specific cleavage ladder wasgenerated by incubating RNA in 1 U RNase T1 (Ambion) with 20 mM sodiumcitrate pH 5.0, 1 mM EDTA, 7 M urea, and 3 μg yeast RNA for 25 min at25° C. RNAs were resolved on an 8% denaturing polyacrylamide gel, driedfor 90 min at 70° C., then visualized on an FX phosphorimager (BioRAD).Band quantification was performed using the Quantity One softwarepackage (BioRAD). To account for well loading variability, quantifiedband intensities were normalized to an adjacent band of similarintensity showing negligible theophylline dependence.

Cell culture and transfection. All cells were maintained at 37° C. in a5% CO₂-humidified incubator. HEK293T, HEK293, HeLa, and HEK293TtTA-d2EGFP cells were maintained in minimal essential medium (MEM) alphamedia (Invitrogen) supplemented with 10% fetal bovine serum (FBS)(Invitrogen), whereas MDA-MB-231 cells were maintained in RMPI 1640 withglutamine (Invitrogen) supplemented with 10% FBS. Cells were transfectedone day after seeding using Fugene 6 (Roche) according to themanufacturer's instructions, followed by the immediate addition ofligand. HEK293T tTA-d2EGFP were transfected with shRNA vector (250 ng),whereas cells lacking endogenous GFP were cotransfected with shRNAvector (250 ng) and the pcDNA3.1(+) (Invitrogen) harboring the d2EGFPgene (25 ng) (Clontech). One day post-transfection the media and ligandwere replaced. Transfected cells were collected three dayspost-transfection for flow cytometry analysis.

Cell fluorescence analysis. Three days post-transfection, cells weretrypsinized and subjected to flow cytometry analysis using the Cell LabQuanta SC MPL (Beckman Coulter). Cells were first gated twice for (1)viability as assessed by electronic volume (EV) versus side scatter (SS)and (2) green fluorescence above autofluorescence to remove anonfluorescent subpopulation. Cells were then gated for either low orhigh DsRed-Express fluorescence, representing untransfected ortransfected cells, respectively. To minimize well-to-well variability,the median green fluorescence value of transfected cells were divided bythat of untransfected cells in the same well and reported as GFP(%). Forcells cotransfected with shRNA and GFP plasmids, GFP(%) is the relativeGFP levels when normalized to mean red fluorescence followed bynormalization to cells transfected with the scrambled shRNA. See FIG. 15for representative plots and the corresponding gates for transfected anduntransfected cells.

Estimation of the lower limit of basal expression levels. Our modelasserts that the active conformation sets the greatest knockdown thatcan be achieved by an shRNA switch containing a given aptamer sequencein a specified experimental setup. This level was evaluated for shRNAswitches that preferentially adopt the active conformation throughswitching strand modifications and contain the larger theophylline(average of S5, S7, S9, and S10), smaller theophylline (average of S7,S14, S15) tetracycline (T3), and xanthine (X3) aptamers. See Table I forthe specified shRNA switch sequences.

Modeling and RNA energetic calculations. Calculation of RNA free energyand partition functions were performed using RNAStructure (Mathews etal, 2004). K_(Comp) and the energy difference between inactive andactive conformations are related by the following expression:

$\begin{matrix}{{{\Delta \; G_{model}} = {{{E{()}} - {E{()}}} = {{- N_{A}}k_{B}{T \cdot {\ln \left( K_{Comp} \right)}}}}},} & (3)\end{matrix}$

where N_(A) is Avogadro's number, k_(B) is the Boltzmann constant, and Tis temperature (K). A full description of the model derivation, methodsfor calculating folding energetics, and prediction of the transferfunction for a given shRNA switch sequence are provided below. Equationfits to measure the correlation strength between ΔG_(method) and basalexpression levels were performed by least-squares analysis using thefollowing expression that has the same mathematical form as equation(1):

$\begin{matrix}{{f_{fit} = {1 - {C_{1}\left\lbrack {C_{2} + {\exp \left( {- \frac{\Delta \; G_{method}}{k_{B}N_{A}T}} \right)}} \right\rbrack}^{- C_{3}}}},} & (4)\end{matrix}$

where C₁₋₃ are the fit constants. Table II contains energetic valuescalculated under each method along with experimentally determinedexpression levels.

REFERENCES

-   Abbas-Terki T, Blanco-Bose W, Deglon N, Pralong W, Aebischer    P (2002) Lentiviral-mediated RNA interference. Hum Gene Ther 13:    2197-2201.-   An C I, Trinh V B, Yokobayashi Y (2006) Artificial control of gene    expression in mammalian cells by modulating RNA interference through    aptamer-small molecule interaction. RNA 12: 710-716.-   Bartlett D W, Davis M E (2006) Insights into the kinetics of    siRNA-mediated gene silencing from live-cell and live-animal    bioluminescent imaging. Nucleic Acids Res 34: 322-333.-   Bayer T S, Smolke C D (2005) Programmable ligand-controlled    riboregulators of eukaryotic gene expression. Nat Biotechnol 23:    337-343.-   Berens C, Thain A, Schroeder R (2001) A tetracycline-binding RNA    aptamer. Bioorg Med Chem 9: 2549-2556.-   Buskirk A R, Landrigan A, Liu D R (2004) Engineering a    ligand-dependent RNA transcriptional activator. Chem Biol 11:    1157-1163.-   Croft L J, Lercher M J, Gagen M J, Mattick J S (2003) Is prokaryotic    complexity limited by accelerated growth in regulatory overhead?    Genome Biology 5: P2.-   Danilova L V, Pervouchine D D, Favorov A V, Mironov A A (2006)    RNAKinetics: a web server that models secondary structure kinetics    of an elongating RNA. J Bioinform Comput Biol 4: 589-596.-   Deans T L, Cantor C R, Collins J J (2007) A Tunable Genetic Switch    Based on RNAi and Repressor Proteins for Regulating Gene Expression    in Mammalian Cells. Cell 130: 363-372.-   Desai S K, Gallivan J P (2004) Genetic screens and selections for    small molecules based on a synthetic riboswitch that activates    protein translation. J Am Chem Soc 126: 13247-13254.-   Dueber J E, Mirsky E A, Lim W A (2007) Engineering synthetic    signaling proteins with ultrasensitive input/output control. Nat    Biotechnol 25: 660-662.-   Elowitz M B, Leibler S (2000) A synthetic oscillatory network of    transcriptional regulators. Nature 403: 335-338.-   Flotte T R (2000) Size does matter: overcoming the adeno-associated    virus packaging limit. Respir Res 1: 16-18.-   Gardner T S, Cantor C R, Collins J J (2000) Construction of a    genetic toggle switch in Escherichia coli. Nature 403: 339-342.-   Grate D, Wilson C (2001) Inducible regulation of the S. cerevisiae    cell cycle mediated by an RNA aptamer-ligand complex. Bioorg Med    Chem 9: 2565-2570.-   Grieger J C, Samulski R J (2005) Packaging capacity of    adeno-associated virus serotypes: impact of larger genomes on    infectivity and postentry steps. J Virol 79: 9933-9944.-   Griffiths-Jones S (2004) The microRNA Registry. Nucleic Acids Res    32: D109-111.-   Griffiths-Jones S, Grocock R J, van Dongen S, Bateman A, Enright A    J (2006) miRBase: microRNA sequences, targets and gene nomenclature.    Nucleic Acids Res 34: D140-144.-   Grundy F J, Henkin T M (2006) From ribosome to riboswitch: control    of gene expression in bacteria by RNA structural rearrangements.    Crit. Rev Biochem Mol Biol 41: 329-338.-   Hall B, Hesselberth J R, Ellington A D (2007) Computational    selection of nucleic acid biosensors via a slip structure model.    Biosens Bioelectron 22: 1939-1947.-   Hooshangi S, Thiberge S, Weiss R (2005) Ultrasensitivity and noise    propagation in a synthetic transcriptional cascade. Proc Natl Acad    Sci USA 102: 3581-3586.-   Huang C Y, Ferrell J E, Jr. (1996) Ultrasensitivity in the    mitogen-activated protein kinase cascade. Proc Natl Acad Sci USA 93:    10078-10083.-   Hutvagner G, Simard M J, Mello C C, Zamore P D (2004)    Sequence-specific inhibition of small RNA function. PLoS Biol 2:    E98.-   Isaacs F J, Dwyer D J, Collins J J (2006) RNA synthetic biology. Nat    Biotechnol 24: 545-554.-   Isaacs F J, Dwyer D J, Ding C, Pervouchine D D, Cantor C R, Collins    J J (2004) Engineered riboregulators enable post-transcriptional    control of gene expression. Nat Biotechnol 22: 841-847.-   Jenison R D, Gill S C, Pardi A, Polisky B (1994) High-resolution    molecular discrimination by RNA. Science 263: 1425-1429.-   Kiga D, Futamura Y, Sakamoto K, Yokoyama S (1998) An RNA aptamer to    the xanthine/guanine base with a distinctive mode of purine    recognition. Nucleic Acids Res 26: 1755-1760.-   Koch A L (1956) The metabolism of methylpurines by Escherichia    coli. I. Tracer studies. J Biol Chem 219: 181-188.-   Lee J F, Hesselberth J R, Meyers L A, Ellington A D (2004) Aptamer    database. Nucleic Acids Res 32: D95-100.-   Lynch S A, Desai S K, Sajja H K, Gallivan J P (2007) A    high-throughput screen for synthetic riboswitches reveals    mechanistic insights into their function. Chem Biol 14: 173-184.-   Macrae I J, Zhou K, Li F, Repic A, Brooks A N, Cande W Z, Adams P D,    Doudna J A (2006) Structural basis for double-stranded RNA    processing by Dicer. Science 311: 195-198.-   Malphettes L, Fussenegger M (2006) Impact of RNA interference on    gene networks. Metab Eng 8: 672-683.-   Mathews D H, Disney M D, Childs J L, Schroeder S J, Zuker M, Turner    D H (2004) Incorporating chemical modification constraints into a    dynamic programming algorithm for prediction of RNA secondary    structure. Proc Natl Acad Sci USA 101: 7287-7292.-   Meister G, Landthaler M, Dorsett Y, Tuschl T (2004)    Sequence-specific inhibition of microRNA- and siRNA-induced RNA    silencing. RNA 10: 544-550.-   Ogawa A, Maeda M (2008) An artificial aptazyme-based riboswitch and    its cascading system in E. coli. Chembiochem 9: 206-209.-   Parisien M, Major F (2008) The MC-Fold and MC-Sym pipeline infers    RNA structure from sequence data. Nature 452: 51-55.-   Raab R M, Stephanopoulos G (2004) Dynamics of gene silencing by RNA    interference. Biotechnol Bioeng 88: 121-132.-   Rinaudo K, Bleris L, Maddamsetti R, Subramanian S, Weiss R, Benenson    Y (2007) A universal RNAi-based logic evaluator that operates in    mammalian cells. Nat. Biotechnol.-   Seelig G, Soloveichik D, Zhang D Y, Winfree E (2006) Enzyme-free    nucleic acid logic circuits. Science 314: 1585-1588.-   Shalgi R, Lieber D, Oren M, Pilpel Y (2007) Global and Local    Architecture of the Mammalian microRNA-Transcription Factor    Regulatory Network. PLoS Comput Biol 3: e131.-   Soukup G A, Breaker R R (1999) Relationship between internucleotide    linkage geometry and the stability of RNA. RNA 5: 1308-1325.-   Stojanovic M N, Stefanovic D (2003) A deoxyribozyme-based molecular    automaton. Nat Biotechnol 21: 1069-1074.-   Sudarsan N, Barrick J E, Breaker R R (2003) Metabolite-binding RNA    domains are present in the genes of eukaryotes. RNA 9: 644-647.-   Suel G M, Kulkarni R P, Dworkin J, Garcia-Ojalvo J, Elowitz M    B (2007) Tunability and noise dependence in differentiation    dynamics. Science 315: 1716-1719.-   Suess B, Hanson S, Berens C, Fink B, Schroeder R, Hillen W (2003)    Conditional gene expression by controlling translation with    tetracycline-binding aptamers. Nucleic Acids Res 31: 1853-1858.-   Suess B, Weigand J E (2008) Engineered riboswitches—Overview,    Problems and Trends. RNA Biol 5.-   Thompson K M, Syrett H A, Knudsen S M, Ellington A D (2002) Group I    aptazymes as genetic regulatory switches. BMC Biotechnol 2: 21.-   Weigand J E, Suess B (2007) Tetracycline aptamer-controlled    regulation of pre-mRNA splicing in yeast. Nucleic Acids Res 35:    4179-4185.-   Wieland M, Hartig J S (2008) Improved aptazyme design and in vivo    screening enable riboswitching in bacteria. Angew Chem Int Ed Engl    47: 2604-2607.-   Win M N, Smolke C D (2007) From the Cover: A modular and extensible    RNA-based gene-regulatory platform for engineering cellular    function. Proc Natl Acad Sci USA 104: 14283-14288.-   Yokobayashi Y, Weiss R, Arnold F H (2002) Directed evolution of a    genetic circuit. Proc Natl Acad Sci USA 99: 16587-16591.-   Zeng Y, Cullen B R (2004) Structural requirements for pre-microRNA    binding and nuclear export by Exportin 5. Nucleic Acids Res 32:    4776-4785.-   Zimmermann G R, Wick C L, Shields T P, Jenison R D, Pardi A (2000)    Molecular interactions and metal binding in the theophylline-binding    core of an RNA aptamer. RNA 6: 659-667.-   Win and Smolke, Proc Natl Acad Sci USA. 104(36): 14283-14288, 2007.

All reference cited herein, including any and all associated supportingonline materials (if applicable), are incorporated herein by reference.

1. A polynucleotide comprising: (1) a modular actuator domain having oneor more functional activities, wherein said modular actuator domain isnot a ribozyme, (2) a modular sensor domain that detects concentrationchange of a molecule, or status change of an environmental condition(pH, ion concentration, temperature), and, (3) an informationtransmission domain between the modular actuator domain and the modularsensor domain, said information transmission domain comprising: (a) ageneral transmission region, (b) a switching strand, (c) a competingstrand, wherein the switching strand and the competing strand are in acontinuous sequence and compete to bind to the general transmissionregion through hybridization interactions; wherein detection of theconcentration or status change by the modular sensor domain favors aconformation change in the modular actuator domain; wherein saidconformation change is mediated by a strand-displacement mechanism inthe information transmission domain to favor the binding of the generaltransmission region to one of said switching strand and said competingstrand; and, wherein said conformation change modulates said functionalactivities.
 2. The polynucleotide of claim 1, which is an RNA, a DNA, ora combination thereof.
 3. The polynucleotide of claim 1, wherein saidmodular actuator domain comprises an antisense sequence, an siRNA orprecursor thereof, an miRNA or precursor thereof, an shRNA or precursorthereof, an RNase III substrate, an alternative splicing element, or anRNAi targeting sequence.
 4. The polynucleotide of claim 1, wherein saidfunctional activities comprise an ability to hybridize with a targetpolynucleotide, an ability to be incorporated into a RISC complex toserve as an siRNA or miRNA guide sequence, or an ability to be an RNaseIII substrate.
 5. The polynucleotide of claim 1, wherein said modularsensor domain is an aptamer.
 6. The polynucleotide of claim 1, whereinsaid modular sensor domain binds the molecule, preferably specifically.7. The polynucleotide of claim 1, wherein the switching strand and thecompeting strand substantially do not overlap.
 8. The polynucleotide ofclaim 1, wherein the switching strand and the competing strand havesubstantially the same sequence.
 9. The polynucleotide of claim 1,wherein the switching strand and the competing strand are in tandem. 10.The polynucleotide of claim 1, wherein said conformation changeincreases said functional activities.
 11. The polynucleotide of claim 1,wherein the extent of the conformation change is amenable to (rational)adjustment/tuning.
 12. The polynucleotide of claim 11, wherein saidadjustment/tuning is effectuated by modifying base-pairing interactionsamong the general transmission region, the switching strand, and/or thecompeting strand.
 13. The polynucleotide of claim 12, wherein saidadjustment/tuning is effectuated by changing the length of the paringbase-pairs at one or both ends of the duplex formed between the generaltransmission region and the switching strand, and/or the duplex formedbetween the general transmission region and the competing strand. 14.The polynucleotide of claim 12, wherein said adjustment/tuning iseffectuated by changing base-pairing complementarity.
 15. Thepolynucleotide of claim 11, wherein said adjustment/tuning iseffectuated by changing the binding affinity between the modular sensordomain and the molecule without changing the size of the modular sensordomain.
 16. The polynucleotide of claim 11, wherein saidadjustment/tuning is effectuated by changing the size of the modularsensor domain.
 17. A method for rational design of a modularpolynucleotide, the method comprising: (1) providing a modular actuatordomain having one or more functional activities, wherein said modularactuator domain is not a ribozyme, (2) providing a modular sensor domainthat detects concentration change of a molecule, or status change of anenvironmental condition (pH, ion concentration, temperature), and, (3)providing an information transmission domain between the modularactuator domain and the modular sensor domain, said informationtransmission domain comprising: (a) a general transmission region, (b) aswitching strand, (c) a competing strand, wherein the switching strandand the competing strand are in a continuous sequence and compete tobind to the general transmission region through hybridizationinteractions, wherein detection of the concentration or status change bythe modular sensor domain favors a conformation change in the modularactuator domain; wherein said conformation change is mediated by astrand-displacement mechanism in the information transmission domain tofavor the binding of the general transmission region to one of saidswitching strand and said competing strand; and, wherein saidconformation change modulates said functional activities.
 18. A methodfor improving the design of a sensor-regulated polynucleotide, saidpolynucleotide comprising: (1) an actuator domain having one or morefunctional activities, wherein said actuator domain is not a ribozyme,and, (2) a sensor domain that detects concentration change of amolecule, or status change of an environmental condition (pH, ionconcentration, temperature), the method comprising: providing aninformation transmission domain between the actuator domain and thesensor domain, said information transmission domain comprising: (a) ageneral transmission region, (b) a switching strand, (c) a competingstrand, wherein the switching strand and the competing strand are in acontinuous sequence and compete to bind to the general transmissionregion through hybridization interactions; wherein detection of theconcentration or status change by the sensor domain favors aconformation change in the actuator domain; wherein said conformationchange is mediated by a strand-displacement mechanism in the informationtransmission domain to favor the binding of the general transmissionregion to one of said switching strand and said competing strand; and,wherein said conformation change modulates said functional activities.19. A vector or expression construct encoding the polynucleotide ofclaim
 1. 20. The vector or expression construct of claim 19, furthercomprising one or more transcriptional regulatory sequences thatregulate transcription from said vector or expression construct in acell containing said vector or expression construct.
 21. A cellengineered to include the polynucleotide of claim 1, or the vector orexpression construct of claim
 19. 22. A method for regulating expressionof a recombinant gene, comprising: (i) providing a cell of claim 21,(ii) contacting the cell with said molecule in an amount that alters theactivity of said modular actuator domain.
 23. A cell comprising: ametabolic pathway of one or more reactions that are regulated at leastin part by a target gene; and one or more polynucleotide of claim 1 thatact as control elements on said metabolic pathway by regulatingexpression of said target gene through said modular actuator domain,wherein binding of said molecule to said modular sensor domain (e.g.,aptamer) causes a change in the intramolecular interaction of saidinformation transmission domain, such that there is a change in theregulation of said target gene by said modular actuator domain, at arate dependent upon the presence or absence of said molecule.
 24. Thecell of claim 23, wherein the metabolic pathway includes at least onereaction mediated by an enzyme, and at least one of said polynucleotideregulates expression of said enzyme.
 25. The cell of claim 23, whereinsaid modular actuator domain comprising a substrate for RNase III,wherein said substrate, when processed by RNase III, produces an siRNAor miRNA that targets a transcript of said target gene.
 26. A method forrendering expression of a target gene in a cell dependent on thepresence or absence of a molecule, comprising introducing into the cella polynucleotide of claim 1 comprising a modular actuator domaincomprising a substrate for RNase III, wherein said substrate, whenprocessed by RNase III, produces an siRNA or miRNA that targets atranscript of said target gene, wherein, binding of said molecule tosaid modular sensor domain (e.g., aptamer) causes a change in theintramolecular interaction of said information transmission domain, suchthat said substrate is processed by RNase III to produce the siRNA ormiRNA to target said transcript, at a rate dependent upon the presenceor absence of said molecule.
 27. A method of determining the amount ofan analyte in a cell which expresses a reporter gene, comprising: (1)introducing into the cell a polynucleotide of claim 1 comprising amodular actuator domain comprising a substrate for RNase III, whereinsaid substrate, when processed by RNase III, produces an siRNA or miRNAthat targets a transcript of said reporter gene, wherein binding of saidanalyte to said modular sensor domain (e.g., aptamer) causes a change inthe intramolecular interaction of said information transmission domain,such that said substrate is processed by RNase III to produce said siRNAor miRNA to inhibit expression of said reporter gene, at a ratedependent upon the presence or absence of said analyte; (2) measuringthe amount of expression of said reporter gene; and (3) correlating theamount of expression of said reporter gene with the amount of analyte,thereby determining the amount of the analyte in the cell.
 28. A methodfor treating or preventing infection by a pathogenic agent, comprisingadministering to a patient a sufficient amount of a polynucleotide ofclaim 1, wherein said molecule is produced as a consequence of infectionby said pathogenic agent, and wherein said modular actuator domaininhibits the function of one or more genes essential for successfulinfection by said pathogenic agent.
 29. A method for causing phenotypicregulation of cell growth, differentiation or viability in cells of apatient, comprising introducing into cells in said patient apolynucleotide of claim 1, where said modular sensor domain (e.g.,aptamer) binds to the molecule, the concentration of which is dependenton cellular phenotype, wherein binding of said molecule to said modularsensor domain favors a conformational change that increases (ordecreases) said functional activities of said modular actuator domain,and said increased or decreased functional activities of said modularactuator domain modulates expression of a target gene essential foraltering the regulation of cell growth, differentiation or viability insaid cells.
 30. A pharmaceutical preparation comprising a polynucleotideof claim 1, or an expression construct which, when transcribed, producesan RNA including said polynucleotide, and a pharmaceutically acceptablecarrier suitable for use administration to a human or non-human patient.